Single probe, multiple temperature, nucleic acid detection methods, kits, and compositions

ABSTRACT

Provided herein are methods, kits, and compositions related to nucleic acid detection assays that allow discrimination of multiple target sequences with a single probe. In particular, provided herein are methods kits, and compositions that include single-probe target sequence discrimination where different target amplicons may have identical probe hybridization sequences by employing multiple temperature end-point signal probe detection. Also provided herein are methods, kits, and compositions for distinguishing between two or more target amplicons using multiple-temperature end-point probe detection. In certain embodiments, asymmetric PCR amplification methods are employed (e.g., LATE-PCR amplification).

The present application claims priority to U.S. Provisional PatentApplication Ser. No. 61/232,999, filed Aug. 11, 2009, the entiredisclosure of which is herein incorporated by reference in its entirety.

FIELD

Provided herein are methods, kits, and compositions related to nucleicacid detection assays that allow discrimination of multiple targetsequences with a single probe. In particular, provided herein aremethods kits, and compositions that include single-probe target sequencediscrimination where different target amplicons may have identical probehybridization sequences by employing multiple temperature end-pointsignal probe detection. Also provided herein are methods, kits, andcompositions for distinguishing between two or more target ampliconsusing multiple-temperature end-point probe detection. In certainembodiments, asymmetric PCR amplification methods are employed (e.g.,LATE-PCR amplification).

BACKGROUND

Staphylococcus aureus (S. aureus) can be a virulent pathogen of animalsand humans. It can also cause severe food poisoning by the production ofa toxin. Diseases caused by S. aureus cover a very wide clinicalspectrum, from simple skin infections to life threatening infections ofthe bones, heart, and organs. Of particular concern is the recognitionthat S. aureus infection is common after surgery. It is also associatedwith intravenous tubing and other implants.

The bacterium S. aureus may be transmitted between healthy individualsby skin to skin contact, or from a commonly shared item or a surface(e.g., tanning beds, gym equipment, food handling equipment, etc.) wherethe transfer may be made to a subsequent person who uses the shared itemor touches the surface. Of great medical concern is the recognition thathealthy people entering hospitals may “carry” S. aureus (e.g., on theirskin, in their noses, etc.) without any signs or symptoms. In thepresence of favorable conditions (often found in, but not limited tohospitals), the S. aureus can activate and cause serious infection. Inaddition, S. aureus can also be a source of food poisoning, often causedby a food handler contaminating the food product.

There are two broad categories of S. aureus based on an individualclone's susceptibility to a class of B. lactam antibiotics that includesmethicillin. These are methicillin susceptible S. aureus (MSSA), andmethicillin resistant S. aureus (MRSA). Until only a few years ago,various strains of MRSA were almost exclusively found in hospitals. Now,many are also present in the noses, skin, etc. of people in thenon-hospital community. Moreover, these MRSA strains are increasinglycausing serious infections in the community. MRSA is particularlyserious because very few antibiotics (e.g., vancomycin) have been shownto be uniformly effective against MRSA.

The Center for Disease Control and Prevention actively surveys for thedevelopment of methicillin resistant S. aureus. In 2000, the Society forHealthcare Epidemiology of America guidelines recommended contactisolation for patients with MRSA. In addition to the morbidity andmortality caused by MRSA, it has been estimated that each case ofinfection costs at least $23,000. Methicillin-susceptible Staphylococcusaureus (MSSA) and Methicillin-resistant Staphylococcus aureus (MRSA) areincreasingly infecting people worldwide in both hospitals and within thecommunity. The rate of infections in ICUs is especially troubling,rising from 2% in 1974 to 64% in 2004. One in three people carry MSSAwhile one in 100 people carry MRSA.

SUMMARY

Provided herein are methods, kits, and compositions related to nucleicacid detection assays that allow discrimination of multiple targetsequences with a single probe. In particular, provided herein aremethods kits, and compositions that include single-probe target sequencediscrimination where different target amplicons may have identical probehybridization sequences by employing multiple temperature end-pointsignal probe detection. Also provided herein are methods, kits, andcompositions for distinguishing between two or more target ampliconsusing multiple-temperature end-point probe detection. In certainembodiments, asymmetric PCR amplification methods are employed (e.g.,LATE-PCR amplification).

In some embodiments, methods are provided for identifying the presenceof polymorphic target sequence variants (e.g., SCCmec variants) in asample, comprising: a) providing: i) a sample suspected of containing: afirst or second variant of a polymorphic target sequence, ii) a labeledprobe, iii) a first variant temperature/temperature signal ratio, iv) asecond variant temperature/temperature signal ratio, v) at least oneforward primer, and vi) at least one reverse primer; b) combining thesample, the labeled probe, the forward primer, and the reverse primer togenerate a combined sample and treating the combined sample underamplification conditions such that: a first single-stranded amplicon isgenerated if the first variant is present, and a second single-strandedamplicon is generated if the second variant is present, wherein thefirst and second single-stranded amplicons each comprise the followingidentical sequences: i) a probe hybridization sequence, ii) a 5′ endcorresponding to the sequence of the reverse primer, and iii) a 3′ endcomplementary to the forward primer; and wherein the first and secondsingle-stranded amplicons do not have complete sequence identity; c)exposing the combined sample to multiple temperatures that allow thelabeled probe to hybridize to the probe hybridization sequence andproduce temperature-dependent signals; d) detecting thetemperature-dependent signals at least two temperatures; e) generatingan experimental temperature/temperature signal ratio; and f) comparingthe experimental temperature/temperature signal ratio with the first andsecond variant temperature/temperature signal ratios, wherein a matchbetween the experimental temperature/temperature signal ratio and thefirst or second variant temperature/temperature signal ratio identifiesthe presence of the first or second variant in the sample.

In certain embodiments, the forward primer comprises a limiting primerand the reverse primer comprises an excess primer, wherein the excessprimer is added to the combined sample at a concentration at leasttwo-times (e.g., two-times . . . five-times . . . 50-times . . .100-times . . . 1000-times . . . ) that of the limiting primer, andwherein the amplification conditions comprise asymmetric PCR conditions.In some embodiments, the forward primer comprises an excess primer andthe reverse primer comprises a limiting primer, wherein the excessprimer is added to the combined sample at a concentration at leasttwo-times (e.g., two-time . . . five-times . . . 50-times . . .100-times . . . 1000-times . . . ) that of the limiting primer, andwherein the amplification conditions comprise asymmetric PCR conditions.In other embodiments, the asymmetric PCR conditions are LATE-PCRconditions, and wherein the initial melting temperature of the limitingprimer is equal to or higher than the initial melting temperature of theexcess primer. In further embodiments, each of the single-strandedamplicons comprise at least one amplicon spacer region selected from: A)a 5′ spacer region that is adjacent to the 5′ end and the probehybridization site, and B) a 3′ spacer regions adjacent to the 3′ endand the probe hybridization site; and wherein at least one of the 5′ and3′ spacer regions differ in sequence between the first and secondsingle-stranded amplicons. In particular embodiments, the 5′ and/or 3′spacer regions have secondary structure within a temperature range25-85° C.

In some embodiments, the presence of the first or second variant isidentified in the sample by finding a match between the experimentaltemperature/temperature signal ratio and the first or second varianttemperature/temperature signal ratio. In other embodiments, the methodsfurther comprise: providing a combined-variant temperature/temperaturesignal ratio, and comparing the experimental temperature/temperaturesignal ratio to the combined-variant temperature/temperature signalratio, wherein a match between the experimental temperature/temperaturesignal ratio and the combined-variant temperature/temperature signalratio identifies the presence of both the first and second variants inthe sample. In particular embodiments, the presence of the first andsecond variants is identified in the sample by finding a match betweenthe experimental temperature/temperature signal ratio and thecombined-variant temperature/temperature signal ratio. In otherembodiments, a match is found when the experimentaltemperature/temperature signal ratio is within 0.4 (e.g., 0.4, 0.3, 0.2,0.1, or 0.0) of the first or second variant temperature/temperaturesignal ratios.

In further embodiments, the exposing the combined sample to multipletemperatures comprises gradually cooling the combined sample such thatthe temperature-dependent signals are binding signals. In additionalembodiments, the exposing the combined sample to multiple temperaturescomprises gradually heating the combined sample such that thetemperature-dependent signals are melting signals. In certainembodiments, the gradual cooling or the gradual heating is carried outmore than once and the values of these repeats are averaged.

In further embodiments, the generating an experimentaltemperature/temperature signal ratio includes normalizing thetemperature-dependent signals with a reference temperature, and whereinthe first and second variant temperature/temperature signal ratios arenormalized with the reference temperature.

In particular embodiments, the labeled probe comprises a molecularbeacon probe. In certain embodiments, the molecular beacon probecomprises a stem that is precisely two base-pairs in length.

In some embodiments, the polymorphic target sequences comprises a SCCmecregion from MRSA spanning the mecA-orfX boundary, and wherein the firstand second single-stranded amplicons each comprise a portion of the mecAgene and a portion of the orfX region. In other embodiments, the firstor second variants are selected from: SCCmec type I, SCCmec type II,SCCmec type III, SCCmec type IV, SCCmec type V, and SCCmec type VII.

In some embodiments, compositions are provided comprising: a) a labeledprobe; b) a forward primer; c) a reverse primer; d) first and secondsingle-stranded amplicons that each comprise the following identicalsequences: i) a probe hybridization sequence, ii) a 5′ end correspondingto the sequence of the reverse primer, and iii) a 3′ end complementaryto the forward primer; and wherein the first and second single-strandedamplicons do not have complete sequence identity (e.g., they differ by 1base, or 2 bases . . . 5 bases . . . or more).

In certain embodiments, systems are provided comprising: a) a labeledprobe; b) a first variant temperature/temperature signal ratio; c) asecond variant temperature/temperature signal ratio; d) a forwardprimer; e) a reverse primer; f) first and second single-strandedamplicons that each comprise the following identical sequences: i) aprobe hybridization sequence, ii) a 5′ end corresponding to the sequenceof the reverse primer, and iii) a 3′ end complementary to the forwardprimer; and wherein the first and second single-stranded amplicons donot have complete sequence identity.

In some embodiments, methods are provided for identifying the presenceof polymorphic target sequence variants (e.g., SCCmec variants) in asample comprising: a) providing: i) a sample suspected of containing: afirst or second variant of a polymorphic target sequence, ii) a labeledprobe, iii) a first variant temperature/temperature signal ratio, iv) asecond variant temperature/temperature signal ratio, v) first and secondforward primers that differ in sequence, and vi) a reverse primer; b)combining the sample, the labeled probe, the first and second forwardprimers, and the reverse primer to generate a combined sample andtreating the combined sample under amplification conditions such that: afirst single-stranded amplicon is generated comprising a first 3′ endcomplementary to the first forward primer, and a second single-strandedamplicon is generated comprising a second 3′ end complementary to thesecond forward primer, and wherein the first and second single-strandedamplicons each further comprise the following identical sequences: i) aprobe hybridization sequence, and ii) a 5′ end corresponding to thesequence of the reverse primer; c) exposing the combined sample tomultiple temperatures that allow the labeled probe to hybridize to theprobe hybridization sequence and produce temperature-dependent signals;d) detecting the temperature-dependent signals at least twotemperatures; e) generating an experimental temperature/temperaturesignal ratio; and f) comparing the experimental temperature/temperaturesignal ratio with the first and second variant temperature/temperaturesignal ratios, wherein a match between the experimentaltemperature/temperature signal ratio and the first or second varianttemperature/temperature signal ratios identifies the presence of thefirst or second variant in the sample.

In other embodiments, the first and second forward primers comprisecorresponding first and second limiting primers, and the reverse primercomprises an excess primer, wherein the excess primer is added to thecombined sample at a concentration at least five-times that of the firstand second limiting primers, and wherein the amplification conditionscomprise asymmetric PCR conditions. In further embodiments, the firstsingle-stranded amplicon is generated in an amount that is at least2-fold greater (e.g., 2-fold . . . 10-fold . . . 25-fold . . . 100-fold. . . 1000-fold . . . ) than the second single-stranded amplicon, andwherein there is a match between the experimentaltemperature/temperature signal ratio and the first varianttemperature/temperature signal ratio, thereby identifying the presenceof the first variant. In other embodiments, the second single-strandedamplicon is generated in an amount that is at least 2-fold greater(e.g., 2-fold . . . 10-fold . . . 25-fold . . . 100-fold . . . 1000-fold. . . ) than the first single-stranded amplicon, and wherein there is amatch between the experimental temperature/temperature signal ratio andthe second variant temperature/temperature signal ratio, therebyidentifying the presence of the second variant.

In some embodiments, methods are provided for identifying the presenceof polymorphic target sequence variants (e.g., SCCmec variants) in asample comprising: a) providing: i) a sample suspected of containing: afirst, second, or third variant of a polymorphic target sequence, ii) alabeled probe, iii) a first variant temperature/temperature signalratio, iv) a second variant temperature/temperature signal ratio, v) athird variant temperature/temperature signal ratio, vi) a forwardprimer, and vii) a reverse primer; b) combining the sample, the labeledprobe, the forward primer, and the reverse primer to generate a combinedsample and treating the combined sample under amplification conditionssuch that: a first single-stranded amplicon is generated if the firstvariant is present, a second single-stranded amplicon is generated ifthe second variant is present, and a third-single stranded amplicon isgenerated if the third variant is present, wherein the first, second,and third single-stranded amplicons each comprise the followingidentical sequences: i) a 5′ end corresponding to the sequence of thereverse primer, and ii) a 3′ end complementary to the forward primer;and wherein the first, second, and third single-stranded ampliconscomprise identical or different probe hybridization sequences, and donot have complete sequence identity to each other; c) exposing thecombined sample to multiple temperatures that allow the labeled probe tohybridize to the probe hybridization sequences and producetemperature-dependent signals; d) detecting the temperature-dependentsignals at least two temperatures; e) generating an experimentaltemperature/temperature signal ratio; and f) comparing the experimentaltemperature/temperature signal ratio with the first, second, and thirdvariant temperature/temperature signal ratios, wherein a match betweenthe experimental temperature/temperature signal ratio and the first,second, or third variant temperature/temperature signal ratio identifiesthe presence of the first, second, or third variant in the sample.

In certain embodiments, the at least two of the first, second, and thirdsingle-stranded amplicons comprise identical probe hybridizationsequences. In other embodiments, all three of the first, second, andthird single-stranded amplicons comprise identical probe hybridizationsequences.

In other embodiments, methods are provided for identifying the presenceof polymorphic target sequence variants (e.g., SCCmec variants) in asample comprising: a) providing: i) a sample suspected of containing: afirst, second, or third variant of a polymorphic target sequence, ii) alabeled probe, iii) a first variant temperature/temperature signalratio, iv) a second variant temperature/temperature signal ratio, v) athird variant temperature/temperature signal ratio, vi) first and secondforward primers that differ in sequence, and vii) a reverse primer; b)combining the sample, the labeled probe, the first and second forwardprimers, and the reverse primer to generate a combined sample andtreating the combined sample under amplification conditions such that: afirst single-stranded amplicon is generated comprising a first 3′ endcomplementary to the first forward primer, a second single-strandedamplicon is generated comprising a second 3′ end complementary to thesecond forward primer, and a third single-stranded amplicon is generatedcomprising a third 3′ end that is complementary to either the firstforward primer or the second forward primer, and wherein the first,second, and third single-stranded amplicons each further comprise: i)identical 5′ ends corresponding to the sequence of the reverse primer,and ii) identical or different probe hybridization sequences; c)exposing the combined sample to multiple temperatures that allow thelabeled probe to hybridize to the probe hybridization sequences andproduce temperature-dependent signals; d) detecting thetemperature-dependent signals at least two temperatures; e) generatingan experimental temperature/temperature signal ratio; and f) comparingthe experimental temperature/temperature signal ratio with the first,second, and third variant temperature/temperature signal ratios, whereina match between the experimental temperature/temperature signal ratioand the first, second, or third variant temperature/temperature signalratios identifies the presence of the first, second, or third variant inthe sample.

In some embodiments, methods are provided for identifying the SCCmectype present in a sample comprising: a) providing: i) a sample suspectedof containing a SCCmec containing Staphylococcus, including but notlimited to type I, type II, type III, type IV, type V, or type VIISCCmec, ii) a labeled probe, iii) temperature/temperature signal ratio(e.g., in a database) for at least one type from the list: type I, typeII, type III, type IV, type V, and type VII, iv) first, second, andthird forward primers that differ in sequence, and v) a reverse primer;b) combining the sample, the labeled probe, the first, second, and thirdforward primers, and the reverse primer to generate a combined sampleand treating the combined sample under amplification conditions suchthat: type I, II, III, IV, V, and VII single-stranded amplicons aregenerated if the corresponding SSCmec types are present in the sample,wherein: the type I single-stranded amplicon comprises a first 3′ endcomplementary to the first forward primer, the type II, IV, and Vsingle-stranded amplicons each comprise identical second 3′ endscomplementary to the second forward primer, the type III and VIIsingle-stranded amplicons each comprise identical third 3′ endscomplementary to the third forward primer, wherein the type I, II, III,IV, V, and VII single-stranded amplicons each further comprise: i)identical 5′ ends corresponding to the sequence of the reverse primer,and ii) identical or different probe hybridization sequences; andwherein the type I, II, III, IV, V, and VII single-stranded amplicons donot have complete sequence identity; c) exposing the combined sample tomultiple temperatures that allow the labeled probe to hybridize to theprobe hybridization sequences and produce temperature-dependent signals;d) detecting the temperature-dependent signals at least twotemperatures; e) generating an experimental temperature/temperaturesignal ratio; and f) comparing the experimental temperature/temperaturesignal ratio with the type I, II, III, IV, V, and VIItemperature/temperature signal ratios, wherein a match between theexperimental temperature/temperature signal ratio and the type I, II,III, IV, V, or VII temperature/temperature signal ratios identifies thepresence of the type I, II, III, IV, V, or VII SCCmec containingStaphylococcus in the sample.

In particular embodiments, the type I, III, IV, and V single-strandedamplicons comprise identical probe hybridization sequences with respectto the labeled probe. In other embodiments, the type II and VIIsingle-stranded amplicons comprise identical probe hybridizationsequences with respect to the labeled probe. In other embodiments, theforward primers comprises limiting primers and the reverse primercomprises an excess primer, wherein the excess primer is added to thecombined sample at a concentration at least five-times (e.g., 5-times .. . 100-times . . . 1000-times . . . ) that of any of the limitingprimers, and wherein the amplification conditions comprise asymmetricPCR conditions. In particular embodiments, the asymmetric PCR conditionsare LATE-PCR conditions, and wherein the initial melting temperature ofthe limiting primers are equal to or higher than the initial meltingtemperature of the excess primer.

In certain embodiments, each of the single-stranded amplicons compriseat least one amplicon spacer region selected from: A) a 5′ spacer regionthat is adjacent to the 5′ end and the probe hybridization site, and B)a 3′ spacer regions adjacent to the 3′ end and the probe hybridizationsite; and wherein at least one of the 5′ and 3′ spacer regions differ insequence between the type IV and type V single-stranded amplicons.

In other embodiments, the presence of the type I, II, III, IV, V, or VIISCCmec containing Staphylococcus is identified in the sample by findinga match between the experimental temperature/temperature signal ratioand the type I, type II, type III, type IV, type V, and type VIItemperature/temperature signal ratios. In further embodiments, a matchis found when the experimental temperature/temperature signal ratio iswithin 0.4 (e.g., 0.4, 0.3, 0.2, 0.1, and 0.0) of the type I, type II,type III, type IV, type V, and type VII temperature/temperature signalratios.

In some embodiments, the exposing the combined sample to multipletemperatures comprises gradually cooling the combined sample such thatthe temperature-dependent signals are binding signals. In furtherembodiments, the exposing the combined sample to multiple temperaturescomprises gradually heating the combined sample such that thetemperature-dependent signals are melting signals.

In particular embodiments, the generating an experimentaltemperature/temperature signal ratio includes normalizing thetemperature-dependent signals with a reference temperature, and whereinthe type I, type II, type III, type IV, type V, and type VIItemperature/temperature signal ratios are normalized with the referencetemperature.

In additional embodiments, the labeled probe comprises a molecularbeacon probe. In further embodiments, the molecular beacon probecomprises a stem that is precisely two base-pairs in length. In someembodiments, the third forward primer comprises or consists of thesequence shown in SEQ ID NO:33. In other embodiments, the second forwardprimer comprises or consists of the sequence shown in SEQ ID NO:34. Inadditional embodiments, the first forward primer comprises or consistsof the sequence shown in SEQ ID NO:35. In further embodiments, thereverse primer comprises or consists of the sequence shown in SEQ IDNO:36. In additional embodiments, the labeled probe comprises orconsists of the sequence shown in SEQ ID NO:37. In particularembodiments, the type I single-stranded amplicon comprises or consistsof the complement of the sequence shown in SEQ ID NO:38. In otherembodiments, the type II single-stranded amplicon comprises or consistsof the complement of the sequence shown in SEQ ID NO:39. In particularembodiments, the type III single-stranded amplicon comprises or consistsof the complement of the sequence shown in SEQ ID NO:40. In furtherembodiments, the type IV single-stranded amplicon comprises or consistsof the complement of the sequence shown in SEQ ID NO:41. In additionalembodiments, the type V single-stranded amplicon comprises or consistsof the complement of the sequence shown in SEQ ID NO:42. In otherembodiments, the type VII single-stranded amplicon comprises or consistsof the complement of the sequence shown in SEQ ID NO:43.

In particular embodiments, compositions are provided comprising at leastone purified nucleic acid sequence, wherein the at least one purifiednucleic acid sequence comprises or consists of a sequence selected fromthe group consisting of: SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQID NO:36, and SEQ ID NO:37, or the complement of any one of thesesequences; as well as the complement of any one of these sequences. Incertain embodiments, the compositions further comprise at least twopurified nucleic acid sequences selected from the recited group. Infurther embodiments, the compositions further comprise at least threepurified nucleic acid sequences selected from the recited group.

In some embodiments, compositions are provided comprising at least onepurified nucleic acid sequence, wherein the at least one purifiednucleic acid sequence comprises or consists of a sequence selected fromthe group consisting of: SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQID NO:36, SEQ ID NO:37, the complement of SEQ ID NO:38, the complementof SEQ ID NO:39, the complement of SEQ ID NO:40, the complement of SEQID NO:41, the complement of SEQ ID NO:42, and the complement of SEQ IDNO:43; as well as the complement of any one of these sequences. Infurther embodiments, wherein the composition further comprises at leasttwo or at least three, or at least four, or at least five purifiednucleic acid sequences selected from the recited group.

In some embodiments, methods are provided of detecting a target sequencecomprising: a) providing: i) a forward primer, ii) a reverse primer,iii) a labeled probe, wherein the labeled probe is a molecular beaconwith a stem that is precisely two or three base-pairs in length, iv) asample suspected of containing the target sequence, and v) a targetsequence temperature/temperature signal ratio; b) combining the sample,the labeled probe, the forward primer, and the reverse primer togenerate a combined sample and treating the combined sample underamplification conditions such that a target amplicon is generated if thetarget sequence is present in the sample; c) exposing the combinedsample to multiple temperatures that allow the labeled probe tohybridize to the target amplicon and produce temperature-dependentsignals; d) detecting the temperature-dependent signals at least twotemperatures; e) generating an experimental temperature/temperaturesignal ratio; and f) comparing the experimental temperature/temperaturesignal ratio with the target sequence temperature/temperature signalratio, wherein a match between the experimental temperature/temperaturesignal ratio and the target sequence temperature/temperature signalratio identifies the presence of the target sequence in the sample.

In particular embodiments, the target sequence is selected from thegroup consisting of: mecA, femA-SA, femA-SE, lukF-PV, lukS-PV, VanA, andSCCmec. In other embodiments, the labeled probe consists of a sequenceselected from the group consisting of: SEQ ID NO:4, SEQ ID NO:8, SEQ IDNO:12, SEQ ID NO:16, SEQ ID NO:20, SEQ ID NO:37, and SEQ ID NO:59, orcomplement of any of these sequences. In further embodiments, theforward primer consists of a sequence selected from the group consistingof: SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11, SEQ ID NO:15, SEQ ID NO:19,SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:58, or thecomplement of any of these sequences. In further embodiments, thereverse primer consists of a sequence selected from the group consistingof: SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18,SEQ ID NO:36, and SEQ ID NO:57, or the complement of any of thesesequences. In other embodiments, the target amplicon consists of asequence selected from the group consisting of: SEQ ID NO:1, SEQ IDNO:5, SEQ ID NO:9, SEQ ID NO:13, SEQ ID NO:17, the complement of SEQ IDNO:38, the complement of SEQ ID NO:39, the complement of SEQ ID NO:40,the complement of SEQ ID NO:41, the complement of SEQ ID NO:42, thecomplement of SEQ ID NO:43, and the complement of SEQ ID NO:56; or thecomplement of any of these sequences.

In some embodiments, methods are provided of detecting SCCmec containingStaphylococcus comprising: a) providing: i) a forward primer comprisingor consisting of a sequence selected from the group consisting of: SEQID NO:33, SEQ ID NO:34, and SEQ ID NO:35, ii) a reverse primercomprising or consisting of SEQ ID NO:36, iii) a labeled probe, iv) asample suspected of containing SCCmec containing Staphylococcus; b)combining the sample, the labeled probe, the forward primer, and thereverse primer to generate a combined sample and treating the combinedsample under amplification conditions such that a target amplicon isgenerated if the SCCmec containing Staphylococcus is present in thesample; and c) detecting any signal from the labeled probe therebydetecting the presence or absence of the target amplicon in the combinedsample.

In certain embodiments, the labeled probe comprises or consists of SEQID NO:37. In other embodiments, the labeled probe is a molecular beaconprobe, and wherein the molecular beacon probe comprises a stem that isprecisely two base-pairs in length. In further embodiments, the targetamplicon comprises or consists of the complement of SEQ ID NOs 38-43.

In other embodiments, the forward primer comprises a limiting primer andthe reverse primer comprises an excess primer, wherein the excess primeris added to the combined sample at a concentration at least five-timesthat of the limiting primer, and wherein the amplification conditionscomprise asymmetric PCR conditions. In particular embodiments, thedetecting comprises: i) exposing the combined sample to multipletemperatures that allow the labeled probe to hybridize to the targetamplicon and produce temperature-dependent signals; ii) detecting thetemperature-dependent signals at least two temperatures; iii) generatingan experimental temperature/temperature signal ratio; and iv) comparingthe experimental temperature/temperature signal ratio with a SCCmectemperature/temperature signal ratio, wherein a match between theexperimental temperature/temperature signal ratio and the SCCmectemperature/temperature signal ratio identifies the presence of SCCmeccontaining Staphylococcus in the sample.

In further embodiments, compositions are provided comprising at leastone (e.g., at least one, at least two, at least three, at least four, atleast five, at least six, at least seven, or at least eight) purifiednucleic acid sequence, wherein the at least one purified nucleic acidsequence comprises or consists of a sequence selected from the groupconsisting of: any of SEQ ID NOs:33-37, or the complement of any of SEQID NOs:38-55.

In additional embodiments, kits are provided comprising at least twopurified nucleic acid sequences, wherein the at least two (e.g., atleast two, at least three, at least four, at least five, at least six,at least seven, or at least eight) purified nucleic acid sequences eachcomprises or consist of a sequence selected from the group consistingof: any of SEQ ID NOs:33-37, or the complement of any of SEQ IDNOs:38-55.

In some embodiments, methods are provided of detecting mecA containingStaphylococcus comprising: a) providing: i) a forward primer comprisingor consisting of SEQ ID NO:3, ii) a reverse primer comprising orconsisting of SEQ ID NO:2, iii) a labeled probe, iv) a sample suspectedof containing mecA containing Staphylococcus; b) combining the sample,the labeled probe, the forward primer, and the reverse primer togenerate a combined sample and treating the combined sample underamplification conditions such that a target amplicon is generated if themecA containing Staphylococcus is present in the sample; and c)detecting any signal from the labeled probe thereby detecting thepresence or absence of the target amplicon in the combined sample.

In certain embodiments, the labeled probe comprises or consists of SEQID NO:4. In other embodiments, the labeled probe is a molecular beaconprobe, and wherein the molecular beacon probe comprises a stem that isprecisely three base-pairs in length. In further embodiments, the targetamplicon comprises or consists of SEQ ID NO:1 or complement thereof.

In other embodiments, the forward primer comprises a limiting primer andthe reverse primer comprises an excess primer, wherein the excess primeris added to the combined sample at a concentration at least five-times(e.g., at least 5× . . . 100× . . . 1000× . . . ) that of the limitingprimer, and wherein the amplification conditions comprise asymmetric PCRconditions.

In some embodiments, the detecting step comprises: i) exposing thecombined sample to multiple temperatures that allow the labeled probe tohybridize to the target amplicon and produce temperature-dependentsignals; ii) detecting the temperature-dependent signals at least twotemperatures; iii) generating an experimental temperature/temperaturesignal ratio; and iv) comparing the experimental temperature/temperaturesignal ratio with a mecA temperature/temperature signal ratio, wherein amatch between the experimental temperature/temperature signal ratio andthe mecA temperature/temperature signal ratio identifies the presence ofmecA containing Staphylococcus in the sample.

In particular embodiments, compositions are provided comprising at leastone (e.g., at least one, at least two, at least three, at least four, atleast four, etc) purified nucleic acid sequence, wherein the at leastone purified nucleic acid sequence comprises or consists of a sequenceselected from the group consisting of: any of SEQ ID NOs:1-4 orcomplements thereof.

In other embodiments, kits are provided comprising at least two (e.g.,at least two, at least three, at least four, etc.) purified nucleic acidsequences, wherein the at least two purified nucleic acid sequences eachcomprises or consist of a sequence selected from the group consistingof: any of SEQ ID NOs:1-4 or complements thereof.

In further embodiments, methods are provided of detecting femA-SAcontaining S. aureus comprising: a) providing: i) a forward primercomprising or consisting of SEQ ID NO:7, ii) a reverse primer comprisingor consisting of SEQ ID NO:6, iii) a labeled probe, iv) a samplesuspected of containing femA-SA containing S. aureus; b) combining thesample, the labeled probe, the forward primer, and the reverse primer togenerate a combined sample and treating the combined sample underamplification conditions such that a target amplicon is generated if thefemA-SA containing S. aureus is present in the sample; and c) detectingany signal from the labeled probe thereby detecting the presence orabsence of the target amplicon in the combined sample.

In certain embodiments, the labeled probe comprises or consists of SEQID NO:8. In other embodiments, the labeled probe is a molecular beaconprobe, and wherein the molecular beacon probe comprises a stem that isprecisely two base-pairs in length. In further embodiments, the targetamplicon comprises or consists of SEQ ID NO:5 or complement thereof.

In additional embodiments, the forward primer comprises a limitingprimer and the reverse primer comprises an excess primer, wherein theexcess primer is added to the combined sample at a concentration atleast five-times (e.g., 5× . . . 100× . . . 1000× . . . ) that of thelimiting primer, and wherein the amplification conditions compriseasymmetric PCR conditions.

In further embodiments, the detecting comprises: i) exposing thecombined sample to multiple temperatures that allow the labeled probe tohybridize to the target amplicon and produce temperature-dependentsignals; ii) detecting the temperature-dependent signals at least twotemperatures; iii) generating an experimental temperature/temperaturesignal ratio; and iv) comparing the experimental temperature/temperaturesignal ratio with a femA-SA temperature/temperature signal ratio,wherein a match between the experimental temperature/temperature signalratio and the femA-SA temperature/temperature signal ratio identifiesthe presence of femA-SA containing Staphylococcus in the sample.

In some embodiments, compositions are provided comprising at least one(e.g., at least one, at least two, at least three, or at least four)purified nucleic acid sequence, wherein the at least one purifiednucleic acid sequence comprises or consists of a sequence selected fromthe group consisting of: any of SEQ ID NOs:5-8 or complements thereof.

In other embodiments, kits are provided comprising at least two (e.g.,at least two, at least three, or at least four) purified nucleic acidsequences, wherein the at least two purified nucleic acid sequences eachcomprises or consist of a sequence selected from the group consistingof: any of SEQ ID NOs:1-4 or complements thereof.

In some embodiments, methods are provided of detecting femA-SEcontaining Staphylococcus comprising: a) providing: i) a forward primercomprising or consisting of SEQ ID NO:11, ii) a reverse primercomprising or consisting of SEQ ID NO:10, iii) a labeled probe, iv) asample suspected of containing femA-SE containing Staphylococcus; b)combining the sample, the labeled probe, the forward primer, and thereverse primer to generate a combined sample and treating the combinedsample under amplification conditions such that a target amplicon isgenerated if the femA-SE containing Staphylococcus is present in thesample; and c) detecting any signal from the labeled probe therebydetecting the presence or absence of the target amplicon in the combinedsample.

In certain embodiments, the labeled probe comprises or consists of SEQID NO:12. In some embodiments, the labeled probe is a molecular beaconprobe, and wherein the molecular beacon probe comprises a stem that isprecisely two base-pairs in length. In other embodiments, the targetamplicon comprises or consists of SEQ ID NO:9 or complement thereof.

In particular embodiments, the forward primer comprises a limitingprimer and the reverse primer comprises an excess primer, wherein theexcess primer is added to the combined sample at a concentration atleast five-times (e.g., at least 5× . . . 100× . . . 1000× . . . ) thatof the limiting primer, and wherein the amplification conditionscomprise asymmetric PCR conditions.

In some embodiments, the detecting comprises: i) exposing the combinedsample to multiple temperatures that allow the labeled probe tohybridize to the target amplicon and produce temperature-dependentsignals; ii) detecting the temperature-dependent signals at least twotemperatures; iii) generating an experimental temperature/temperaturesignal ratio; and iv) comparing the experimental temperature/temperaturesignal ratio with a femA-SE temperature/temperature signal ratio,wherein a match between the experimental temperature/temperature signalratio and the femA-SE temperature/temperature signal ratio identifiesthe presence of femA-SE containing Staphylococcus in the sample.

In some embodiments, compositions are provided comprising at least one(e.g., at least one, at least two, at least three, or at least four)purified nucleic acid sequence, wherein the at least one purifiednucleic acid sequence comprises or consists of a sequence selected fromthe group consisting of: any of SEQ ID NOs:9-12 or complements thereof.

In further embodiments, kits are provided comprising at least two (e.g.,at least two, at least three, or at least four) purified nucleic acidsequences, wherein the at least two purified nucleic acid sequences eachcomprises or consist of a sequence selected from the group consistingof: any of SEQ ID NOs:9-12 or complements thereof.

In particular embodiments, methods are provided of detecting lukF-PVcontaining Staphylococcus comprising: a) providing: i) a forward primercomprising or consisting of SEQ ID NO:15, ii) a reverse primercomprising or consisting of SEQ ID NO:14, iii) a labeled probe, iv) asample suspected of containing lukF-PV containing Staphylococcus; b)combining the sample, the labeled probe, the forward primer, and thereverse primer to generate a combined sample and treating the combinedsample under amplification conditions such that a target amplicon isgenerated if the lukF-PV containing Staphylococcus is present in thesample; and c) detecting any signal from the labeled probe therebydetecting the presence or absence of the target amplicon in the combinedsample.

In some embodiments, the labeled probe comprises or consists of SEQ IDNO:16. In other embodiments, the labeled probe is a molecular beaconprobe, and wherein the molecular beacon probe comprises a stem that isprecisely three base-pairs in length. In further embodiments, the targetamplicon comprises or consists of SEQ ID NO:13 or complements thereof.In additional embodiments, the forward primer comprises a limitingprimer and the reverse primer comprises an excess primer, wherein theexcess primer is added to the combined sample at a concentration atleast five-times (e.g., 5× . . . 100× . . . 1000× . . . ) that of thelimiting primer, and wherein the amplification conditions compriseasymmetric PCR conditions.

In additional embodiments, the detecting comprises: i) exposing thecombined sample to multiple temperatures that allow the labeled probe tohybridize to the target amplicon and produce temperature-dependentsignals; ii) detecting the temperature-dependent signals at least twotemperatures; iii) generating an experimental temperature/temperaturesignal ratio; and iv) comparing the experimental temperature/temperaturesignal ratio with a lukF-PV temperature/temperature signal ratio,wherein a match between the experimental temperature/temperature signalratio and the lukF-PV temperature/temperature signal ratio identifiesthe presence of lukF-PV containing Staphylococcus in the sample.

In particular embodiments, compositions are provided comprising at leastone (e.g., at least one, at least two, at least three, or at least four)purified nucleic acid sequence, wherein the at least one purifiednucleic acid sequence comprises or consists of a sequence selected fromthe group consisting of: any of SEQ ID NOs:13-16 or complements thereof.

In other embodiments, kits are provided comprising at least two (e.g.,at least two, at least three, or at least four) purified nucleic acidsequences, wherein the at least two purified nucleic acid sequences eachcomprises or consist of a sequence selected from the group consistingof: any of SEQ ID NOs:13-16 or complements thereof.

In some embodiments, methods are provided of detecting VanA containingStaphylococcus comprising: a) providing: i) a forward primer comprisingor consisting of SEQ ID NO:19, ii) a reverse primer comprising orconsisting of SEQ ID NO:18, iii) a labeled probe, and iv) a samplesuspected of containing VanA containing Staphylococcus; b) combining thesample, the labeled probe, the forward primer, and the reverse primer togenerate a combined sample and treating the combined sample underamplification conditions such that a target amplicon is generated if theVanA containing Staphylococcus is present in the sample; and c)detecting any signal from the labeled probe thereby detecting thepresence or absence of the target amplicon in the combined sample.

In further embodiments, the labeled probe comprises or consists of SEQID NO:20. In other embodiments, the labeled probe is a molecular beaconprobe, and wherein the molecular beacon probe comprises a stem that isprecisely two base-pairs in length. In further embodiments, the targetamplicon comprises or consists of SEQ ID NO:17 or complement thereof.

In some embodiments, the forward primer comprises a limiting primer andthe reverse primer comprises an excess primer, wherein the excess primeris added to the combined sample at a concentration at least five-times(e.g., at least 5× . . . 100× . . . 1000× . . . ) that of the limitingprimer, and wherein the amplification conditions comprise asymmetric PCRconditions.

In other embodiments, the detecting comprises: i) exposing the combinedsample to multiple temperatures that allow the labeled probe tohybridize to the target amplicon and produce temperature-dependentsignals; ii) detecting the temperature-dependent signals at least twotemperatures; iii) generating an experimental temperature/temperaturesignal ratio; and iv) comparing the experimental temperature/temperaturesignal ratio with a VanA temperature/temperature signal ratio, wherein amatch between the experimental temperature/temperature signal ratio andthe VanA temperature/temperature signal ratio identifies the presence ofVanA containing Staphylococcus in the sample.

In additional embodiments, compositions are provided comprising at leastone (e.g., at least one, at least two, at least three, or at least four)purified nucleic acid sequence, wherein the at least one purifiednucleic acid sequence comprises or consists of a sequence selected fromthe group consisting of: any of SEQ ID NOs:17-20 or complements thereof.

In further embodiments, kits are provided comprising at least two (e.g.,at least two, at least three, or at least four) purified nucleic acidsequences, wherein the at least two purified nucleic acid sequences eachcomprises or consist of a sequence selected from the group consistingof: any of SEQ ID NOs:17-20 or complements thereof.

In some embodiments, methods are provided of detecting lukS-PVcontaining Staphylococcus comprising: a) providing: i) a forward primercomprising or consisting of SEQ ID NO:58, ii) a reverse primercomprising or consisting of SEQ ID NO:57, iii) a labeled probe, iv) asample suspected of containing lukS-PV containing Staphylococcus; b)combining the sample, the labeled probe, the forward primer, and thereverse primer to generate a combined sample and treating the combinedsample under amplification conditions such that a target amplicon isgenerated if the lukS-PV containing Staphylococcus is present in thesample; and c) detecting any signal from the labeled probe therebydetecting the presence or absence of the target amplicon in the combinedsample.

In certain embodiments, the labeled probe comprises or consists of SEQID NO:59. In other embodiments, the labeled probe is a molecular beaconprobe, and wherein the molecular beacon probe comprises a stem that isprecisely two base-pairs in length. In further embodiments, the targetamplicon comprises or consists of SEQ ID NO:56 or complements thereof.

In some embodiments, the forward primer comprises a limiting primer andthe reverse primer comprises an excess primer, wherein the excess primeris added to the combined sample at a concentration at least five-times(e.g., 5× . . . 100× . . . 1000× . . . ) that of the limiting primer,and wherein the amplification conditions comprise asymmetric PCRconditions.

In other embodiments, the detecting comprises: i) exposing the combinedsample to multiple temperatures that allow the labeled probe tohybridize to the target amplicon and produce temperature-dependentsignals; ii) detecting the temperature-dependent signals at least twotemperatures; iii) generating an experimental temperature/temperaturesignal ratio; and iv) comparing the experimental temperature/temperaturesignal ratio with a lukS-PV temperature/temperature signal ratio,wherein a match between the experimental temperature/temperature signalratio and the lukS-PV temperature/temperature signal ratio identifiesthe presence of lukS-PV containing Staphylococcus in the sample.

In further embodiments, compositions are provided comprising at leastone (e.g., at least one, at least two, at least three, or at least four)purified nucleic acid sequence, wherein the at least one purifiednucleic acid sequence comprises or consists of a sequence selected fromthe group consisting of: any of SEQ ID NOs:56-59 or complements thereof.

In other embodiments, kits are provided comprising at least two (e.g.,at least two, at least three, or at least four) purified nucleic acidsequences, wherein the at least two purified nucleic acid sequences,respectively, comprises or consist of a sequence selected from the groupconsisting of: any of SEQ ID NOs:56-59 or complements thereof.

In some embodiments, methods are provided of detecting bacteria in asample comprising: a) providing: i) forward primers comprising: a mecAforward primer, a femA-SA forward primer, a femA-SE forward primer, alukF-PV forward primer, a lukS-PV forward primer, and a VanA forwardprimer, ii) reverse primers comprising: a mecA reverse primer, a femA-SAreverse primer, a femA-SE reverse primer, a lukF-PV reverse primer, alukS-PV reverse primer, and a VanA reverse primer, iii) labeled probescomprising: a mecA labeled probe, a femA-SA labeled probe, a femA-SElabeled probe, a lukF-PV labeled probe, a lukS-PV labeled probe, and aVanA labeled probe, iv) a sample suspected of containing a bacteriaselected from S. aureus and S. epidermidis; b) combining the sample, thelabeled probe, the forward primer, and the reverse primer to generate acombined sample and treating the combined sample under amplificationconditions such that: i) a femA-SA amplicon is generated if the S.aureus is present, ii) a femA-SE amplicon is generated if the S.epidermidis is present, iii) a mecA amplicon is generated if thebacteria contains a mecA sequence, iv) a lukS-PV amplicon is generatedif the bacteria contains a lukS-PV sequence, v) a lukF-PV amplicon isgenerated if the bacteria contains a lukF-PV sequence, and vi) a VanAamplicon is generated if the bacteria contains a VanA sequence; and c)detecting any signal from the labeled probes thereby detecting thepresence or absence of one or more of the femA-SA, femA-SE, mecA,lukS-PV, lukF-PV, and VanA amplicons in the combined sample.

In certain embodiments, the detecting further indicates that thebacteria is not present in the sample, or that one or more of thefollowing is present in the sample: MSSA, MSSE, MRSA, MRSE, VRSA, VRSE,and CA-MRSA. In other embodiments, the labeled probes are molecularbeacon probes, and wherein the molecular beacon probes each comprise astem that is precisely two base-pairs in length or precisely threebase-pairs in length.

In further embodiments, the mecA labeled probe comprises or consists ofSEQ ID NO: 4, wherein the femA-SA labeled probe comprises or consists ofSEQ ID NO:8, wherein the femA-SE labeled probe comprises or consists ofSEQ ID NO:12, wherein the lukF-PV labeled probe comprises or consists ofSEQ ID NO:16, wherein the lukS-PV labeled probe comprises or consists ofSEQ ID NO:59, and wherein the VanA labeled probe comprises or consistsof SEQ ID NO:20. In other embodiments, the mecA amplicon comprises orconsists of SEQ ID NO:1, wherein the femA-SA amplicon comprises orconsists of SEQ ID NO:5, wherein the femA-SE amplicon comprises orconsists of SEQ ID NO:9, wherein the lukF-PV amplicon comprises orconsists of SEQ ID NO:13, wherein the lukS-PV amplicon comprises orconsists of SEQ ID NO:56, and wherein the VanA amplicon comprises orconsists of SEQ ID NO:17. In additional embodiments, the mecA forwardprimer comprises or consists of SEQ ID NO:3, wherein the femA-SA forwardprimer comprises or consists of SEQ ID NO:7, wherein the femA-SE forwardprimer comprises or consists of SEQ ID NO:11, wherein the lukF-PVforward primer comprises or consists of SEQ ID NO:15, wherein thelukS-PV forward primer comprises or consists of SEQ ID NO:58, andwherein the VanA forward primer comprises or consists of SEQ ID NO:19.In other embodiments, the mecA reverse primer comprises or consists ofSEQ ID NO:2, wherein the femA-SA reverse primer comprises or consists ofSEQ ID NO:6, wherein the femA-SE reverse primer comprises or consists ofSEQ ID NO:10, wherein the lukF-PV reverse primer comprises or consistsof SEQ ID NO:14, wherein the lukS-PV reverse primer comprises orconsists of SEQ ID NO:57, and wherein the VanA reverse primer comprisesor consists of SEQ ID NO:18.

In other embodiments, the forward primers each comprise a limitingprimer and the reverse primers each comprise an excess primer, whereinthe excess primers are added to the combined sample at a concentrationat least five times (e.g., 5× . . . 25× . . . 100× . . . 1000× . . . )that of the limiting primers, and wherein the amplification conditionscomprise asymmetric PCR conditions.

In some embodiments, the detecting comprises: i) exposing the combinedsample to multiple temperatures that allow the labeled probes tohybridize to their corresponding amplicons and producetemperature-dependent signals; ii) detecting the temperature-dependentsignals at least two temperatures; iii) generating an experimentaltemperature/temperature signal ratio; and iv) comparing the experimentaltemperature/temperature signal ratio with mecA, femA-SA, femA-SE,lukF-PV, lukS-PV, and VanA temperature/temperature signal ratios,wherein a match between the experimental temperature/temperature signalratio and the mecA, femA-SA, femA-SE, lukF-PV, lukS-PV, and VanAtemperature/temperature signal ratios identifies the presence of one ormore of the femA-SA, femA-SE, mecA, lukS-PV, lukF-PV, and VanA ampliconsin the sample.

In other embodiments, the at least two temperature are 50° C. and 35°C., or any other two temperatures between 70° C. and 25° C. (e.g., 25°C. . . . 53° C. . . . 70° C.). In particular embodiments, the forwardprimers further comprise a control forward primer; wherein the reverseprimers further comprise a control reverse primer; and wherein thelabeled probes further comprise a control labeled probe. In additionalembodiments, the control forward primer comprises or consists of SEQ IDNO:23, 27, or 31, wherein the control reverse primer comprises orconsists of SEQ ID NO:22, 26, or 30, and wherein the control probecomprises or consists of SEQ ID NO:24, 28, or 32.

In some embodiments, the lukS-PVL labeled probe is labeled with FAM dye;wherein the femA-SA and the femA-SE labeled probes are labeled with CalOrange dye; wherein the vanA and the lukF-PVL labeled probes are labeledwith Cal Red dye; and wherein the mecA labeled probe is labeled withQuasar dye.

In additional embodiments, the lukS-PVL labeled probe is labeled withFAM dye; wherein the femA-SA and the vanA labeled probes are labeledwith Cal Orange dye; wherein the femA-SE and the lukF-PVL labeled probesare labeled with Cal Red dye; and wherein the mecA labeled probe islabeled with Quasar dye.

In certain embodiments, compositions are provided comprising at leastten (e.g., at least ten . . . at least fifteen . . . or at least twenty)purified nucleic acid sequences, wherein the at least ten purifiednucleic acid sequences comprise or consist of a sequence selected fromthe group consisting of: SEQ ID NOs:2-4, SEQ ID NOs:6-8, SEQ IDNOs:10-12, SEQ ID NOs:14-16, SEQ ID NOs:57-59, SEQ ID NOs: 18-20, andSEQ ID NOs:30-32; or complement to any of these sequences.

In some embodiments, kits are provided comprising at least ten (e.g., atleast ten . . . at least fifteen . . . or at least twenty) purifiednucleic acid sequences, wherein the at least ten purified nucleic acidsequences, respectively, comprise or consist of a sequence selected fromthe group consisting of: SEQ ID NOs:2-4, SEQ ID NOs:6-8, SEQ IDNOs:10-12, SEQ ID NOs:14-16, SEQ ID NOs:57-59, SEQ ID NOs: 18-20, andSEQ ID NOs:30-32, or a complement to any of these sequences.

In further embodiments, methods are provided of detecting bacteria in asample comprising: a) providing: i) forward primers comprising: a mecAforward primer, a femA-SA forward primer, a femA-SE forward primer, alukF-PV forward primer, a VanA forward primer, and first, second, andthird SCCmec forward primers; ii) reverse primers comprising: a mecAreverse primer, a femA-SA reverse primer, a femA-SE reverse primer, alukF-PV reverse primer, a VanA reverse primer, and a SCCmec reverseprimer; iii) labeled probes comprising: a mecA labeled probe, a femA-SAlabeled probe, a femA-SE labeled probe, a lukF-PV labeled probe, a VanAlabeled probe, a plurality of SCCmec labeled probes; iv) a samplesuspected of containing a bacteria selected from S. aureus and S.epidermidis; b) combining the sample, the labeled probe, the forwardprimer, and the reverse primer to generate a combined sample andtreating the combined sample under amplification conditions such that:i) a femA-SA amplicon is generated if the S. aureus is present, ii) afemA-SE amplicon is generated if the S. epidermidis is present, iii) amecA amplicon is generated if the bacteria contains a mecA sequence, iv)a lukF-PV amplicon is generated if the bacteria contains a lukF-PVsequence, v) a VanA amplicon is generated if the bacteria contains aVanA sequence, vi) SCCmec type I, type II, type III, type IV, type V,and/or type VII amplicons are generated if the bacteria contains acorresponding SCCmec type I, type II, type III, type IV, type V, or typeVII sequence; and c) detecting any signal from the labeled probesthereby detecting the presence or absence of one or more of the femA-SA,femA-SE, mecA, lukF-PV, VanA, SSCmec type I, SSCmec type II, SSCmec typeIII, SSCmec type IV, SSCmec type V, and SSCmec type VII amplicons in thecombined sample.

In certain embodiments, the detecting further indicates that thebacteria is not present in the sample, or that one or more of thefollowing is present in the sample: MSSA, MSSE, MRSA, MRSE, VRSA, VRSE,and MRSA community strain. In other embodiments, the labeled probes aremolecular beacon probes, and wherein the molecular beacon probes eachcomprise a stem that is precisely two base-pairs in length or preciselythree base-pairs in length.

In additional embodiments, the mecA labeled probe comprises or consistsof SEQ ID NO: 4, wherein the femA-SA labeled probe comprises or consistsof SEQ ID NO:8, wherein the femA-SE labeled probe comprises or consistsof SEQ ID NO:12, wherein the lukF-PV labeled probe comprises or consistsof SEQ ID NO:16, wherein the VanA labeled probe comprises or consists ofSEQ ID NO:20, and wherein the SCCmec labeled probe comprises or consistsof SEQ ID NO:37. In further embodiments, the mecA amplicon comprises orconsists of SEQ ID NO:1, wherein the femA-SA amplicon comprises orconsists of SEQ ID NO:5, wherein the femA-SE amplicon comprises orconsists of SEQ ID NO:9, wherein the lukF-PV amplicon comprises orconsists of SEQ ID NO:13, wherein the VanA amplicon comprises orconsists of SEQ ID NO:17, wherein the SSCmec type I amplicon comprisesor consists of the complement of SEQ ID NO:38, wherein the SSCmec typeII amplicon comprises or consists of the complement of SEQ ID NO:39,wherein the SSCmec type III amplicon comprises or consists of thecomplement of SEQ ID NO:40, wherein the SSCmec type IV ampliconcomprises or consists of the complement of SEQ ID NO:41, wherein theSSCmec type V amplicon comprises or consists of the complement of SEQ IDNO:42, and wherein the SSCmec type VII amplicon comprises or consists ofthe complement of SEQ ID NO:43.

In further embodiments, the mecA forward primer comprises or consists ofSEQ ID NO:3, wherein the femA-SA forward primer comprises or consists ofSEQ ID NO:7, wherein the femA-SE forward primer comprises or consists ofSEQ ID NO:11, wherein the lukF-PV forward primer comprises or consistsof SEQ ID NO:15, wherein the VanA forward primer comprises or consistsof SEQ ID NO:19, and wherein the first SCCmec forward primer comprisesor consists of SEQ ID NO:33, wherein the second SCCmec forward primercomprises or consists of SEQ ID NO:34, and wherein the third SCCmecforward primer comprises or consists of SEQ ID NO:35.

In other embodiments, the mecA reverse primer comprises or consists ofSEQ ID NO:2, wherein the femA-SA reverse primer comprises or consists ofSEQ ID NO:6, wherein the femA-SE reverse primer comprises or consists ofSEQ ID NO:10, wherein the lukF-PV reverse primer comprises or consistsof SEQ ID NO:14, wherein the VanA reverse primer comprises or consistsof SEQ ID NO:18, and wherein the SCCmec reverse primer comprises orconsists of SEQ ID NO:36.

In particular embodiments, the forward primers each comprise a limitingprimer and the reverse primers each comprise an excess primer, whereinthe excess primers are added to the combined sample at a concentrationat least five-times (e.g., 5× . . . 50× . . . 100× . . . 1000× . . . )that of the limiting primers, and wherein the amplification conditionscomprise asymmetric PCR conditions.

In some embodiments, the detecting comprises: i) exposing the combinedsample to multiple temperatures that allow the labeled probes tohybridize to their corresponding amplicons and producetemperature-dependent signals; ii) detecting the temperature-dependentsignals at least two temperatures; iii) generating an experimentaltemperature/temperature signal ratio; and iv) comparing the experimentaltemperature/temperature signal ratio with mecA, femA-SA, femA-SE,lukF-PV, VanA, SCCmec type I, type II, type III, type IV, type V, andtype VII temperature/temperature signal ratios, wherein a match betweenthe experimental temperature/temperature signal ratio and the mecA,femA-SA, femA-SE, lukF-PV, VanA, SCCmec type I, type II, type III, typeIV, type V, and/or type VII temperature/temperature signal ratiosidentifies the presence of one or more of the femA-SA, femA-SE, mecA,lukF-PV, VanA, SCCmec type I, type II, type III, type IV, type V, and/ortype VII amplicons in the sample.

In particular embodiments, the at least two temperature are: 48° C. and35° C. In other embodiments, the SCCmec probe, the femA-SA probe, thefemA-SE probe, and the mecA probe are all detected at the 48° C. Inadditional embodiments, the SCCmec probe, the vanA probe, and thelukF-PVL probe are all detected at the 35° C. In further embodiments,the at least two temperatures are: 50° C., 45° C., 40° C., and 35° C. Inother embodiments, the SCCmec probe, the femA-SE probe, and the femA-SAprobe are all detected at the 50° C. In further embodiments, the SCCmecprobe is detected at the 45° C. In additional embodiments, the SCCmecprobe is detected at the 40° C. In other embodiments, the vanA probe,the lukF-PVL probe, and the mecA probe are all detected at the 50° C.

In certain embodiments, the forward primers further comprise a controlforward primer; wherein the reverse primers further comprise a controlreverse primer; and wherein the labeled probes further comprise acontrol labeled probe. In particular embodiments, the control forwardprimer comprises or consists of SEQ ID NO:23, 27, or 31, wherein thecontrol reverse primer comprises or consists of SEQ ID NO:22, 26, or 30,and wherein the control probe comprises or consists of SEQ ID NO:24, 28,or 32.

In some embodiments, the SCCmec labeled probe is labeled with FAM dye;wherein the femA-SA and the vanA labeled probes are labeled with CalOrange dye; wherein the famA-SE and the lukF-PVL labeled probes arelabeled with Cal Red dye; and wherein the mecA labeled probe is labeledwith Quasar dye. In further embodiments, a portion of the SCCmec labeledprobes are labeled with FAM dye; wherein a portion of the SCCmec labeledprobes are labeled with Cal orange dye; wherein the vanA labeled probeis labeled with Cal Orange dye; wherein the femA-SE and the lukF-PVLlabeled probes are labeled with Cal Red dye; and wherein the femA-SA andthe mecA labeled probes are labeled with Quasar dye.

In particular embodiments, compositions are provided comprising at leastten (e.g., at least ten . . . at least fifteen . . . or at least twenty)purified nucleic acid sequence, wherein the at least ten purifiednucleic acid sequences comprises or consists of a sequence selected fromthe group consisting of: SEQ ID NOs:2-4, SEQ ID NOs:6-8, SEQ IDNOs:10-12, SEQ ID NOs:14-16, SEQ ID NOs:33-37, SEQ ID NOs: 18-20, andSEQ ID NOs:30-32, or any of the complements thereof.

In some embodiments, kits are provided comprising at least ten (e.g., atleast ten . . . at least fifteen . . . or at least twenty) purifiednucleic acid sequences, wherein the at least ten purified nucleic acidsequences each comprises or consist of a sequence selected from thegroup consisting of: SEQ ID NOs:2-4, SEQ ID NOs:6-8, SEQ ID NOs:10-12,SEQ ID NOs:14-16, SEQ ID NOs:33-37, SEQ ID NOs: 18-20, and SEQ IDNOs:30-32, or any of the complements thereof.

DESCRIPTION OF THE FIGURES

FIG. 1A shows the regions or regions that can be used to distinguishdifferent bacteria types, including S. aureus vs.epidermidis/heamolyticus; MSSA vs. MRSA; hospital acquired vs. communityacquired, and VRSA. FIG. 1B shows 42 possible outcomes that are possiblewith certain embodiments of the Staphylococcus detection assays providedherein.

FIG. 2 shows end point detection results from the exemplary monoplexdetection of FemA-SA described in Example 1. Exemplary raw fluorescencedata is shown in FIG. 2A. Normalized data to 70° C. F/F70 is shown inFIG. 2B. Calculated end-point double ratio [F35/F70]/F50/F70] is shownin FIG. 2C.

FIG. 3 shows anneal curve detection results from the exemplary monoplexdetection of FemA-SA described in Example 1. The anneal raw fluorescencedata is shown in FIG. 3A, the normalized anneal fluorescence data toF70° C. is shown in FIG. 3B, and the calculated anneal ratio (doubleratio): [F35/F70]/F47/F70], is shown in FIG. 3C.

FIG. 4 shows melt curve detection results from the exemplary monoplexdetection of FemA-SA described in Example 1. The anneal raw fluorescencedata is shown in FIG. 4A, the normalized anneal fluorescence data toF70° C. is shown in FIG. 4B, and the calculated anneal ratio (doubleratio): [F35/F70]/F47/F70], is shown in FIG. 4C.

FIGS. 5A-C show (femA-SA, FemA-SE, and MecA respectively) rawfluorescent end point detection results from the exemplary multi-plexdetection of FemA-SA, FemA-SE, and mecA described in Example 1.

FIGS. 6A-C (femA-SA, FemA-SE, and MecA respectively) show normalized endpoint detection results from the exemplary multi-plex detection ofFemA-SA, FemA-SE, and mecA described in Example 1.

FIG. 7 shows calculated end-point double ratios [F35/F70]/F50/F70]results from the exemplary multi-plex detection of FemA-SA, FemA-SE, andmecA described in Example 1.

FIGS. 8A-B (FemA-SE and MecA respectively) show raw fluorescent annealcurve detection results from the exemplary multi-plex detection ofFemA-SA, FemA-SE, and mecA described in Example 1.

FIGS. 9A-C (femA-SA, FemA-SE, and MecA respectively) show normalizedanneal curve detection results from the exemplary multi-plex detectionof FemA-SA, FemA-SE, and mecA described in Example 1.

FIGS. 10A-C (femA-SA, FemA-SE, and MecA respectively) shows calculatedend-point double ratios [F35/F70]/F50/F70] results from the exemplarymulti-plex detection of FemA-SA, FemA-SE, and mecA described in Example1.

FIGS. 11A-C (femA-SA, FemA-SE and MecA respectively) show rawfluorescent melt curve detection results from the exemplary multi-plexdetection of FemA-SA, FemA-SE, and mecA described in Example 1.

FIGS. 12A-C (femA-SA, FemA-SE, and MecA respectively) show normalizedmelt curve detection results from the exemplary multi-plex detection ofFemA-SA, FemA-SE, and mecA described in Example 1.

FIGS. 13A-C (femA-SA, FemA-SE, and MecA respectively) shows calculatedend-point double ratios [F35/F70]/F50/F70] results from the exemplarymulti-plex detection of FemA-SA, FemA-SE, and mecA described in Example1.

FIG. 14 shows an exemplary “MRSA 1” type assay configuration.

FIG. 15 shows results from an exemplary MRSA type 1 assay fromExample 1. FIG. 15A shows full LATE-PCR multiplex results for MSSA andMSSE targets, showing only correct predicted results. FIG. 15B showsfull LATE-PCR multiplex results for MRSA and MRSE targets, showing onlycorrect predicted results. FIG. 15C shows full LATE-PCR multiplexresults for VRSA and VRSE targets, showing only correct predictedresults. FIG. 15D shows full LATE-PCR multiplex results for CA-MRSA andcontrol targets, showing only correct predicted results.

FIG. 16 shows results from an exemplary MRSA type 1 assay fromExample 1. Full LATE-PCR multiplex endpoint ratio fluorescence showinginternal control and mecA targets in shown in FIG. 16A. Full LATE-PCRmultiplex endpoint ratio fluorescence showing lukF-PVL and vanA targetsis shown in FIG. 16B. Full LATE-PCR multiplex endpoint ratiofluorescence showing femA-SA and femA-SE targets is shown in FIG. 16C.Full LATE-PCR multiplex endpoint ratio fluorescence showing externalcontrol and lukS-PVL targets is shown in FIG. 16D.

FIG. 17 shows an exemplary “MRSA 2” type assay configuration.

FIG. 18 shows results from an exemplary MRSA type 2 assay fromExample 1. Full LATE-PCR multiplex endpoint ratio fluorescence showingcontrols and lukS-PVL targets is shown in FIG. 18A. Full LATE-PCRmultiplex endpoint ratio fluorescence showing femA-SA and vanA targetsis shown in FIG. 18B. Full LATE-PCR multiplex endpoint ratiofluorescence showing lukF-PVL and femA-SE targets is shown in FIG. 18C.Full LATE-PCR multiplex endpoint ratio fluorescence showing internalcontrol and mecA targets is shown in FIG. 18D.

FIG. 19 shows an exemplary “MRSA 3” type assay configuration.

FIG. 20 shows results from an exemplary MRSA type 3 assay fromExample 1. FIG. 20A shows full LATE-PCR multiplex endpoint ratiofluorescence showing control targets. FIG. 20B shows full LATE-PCRmultiplex endpoint ratio fluorescence showing MSSA target. FIG. 20Cshows full LATE-PCR multiplex endpoint ratio fluorescence showing MSSEtarget. FIG. 20D shows full LATE-PCR multiplex endpoint ratiofluorescence showing HA-MRSA1 target. FIG. 20E shows full LATE-PCRmultiplex endpoint ratio fluorescence showing HA-MRSA2 target. FIG. 20Fshows full LATE-PCR multiplex endpoint ratio fluorescence showingHA-MRSA3 target. FIG. 20G shows full LATE-PCR multiplex endpoint ratiofluorescence showing HA-MRSA4 target. FIG. 20H shows full LATE-PCRmultiplex endpoint ratio fluorescence showing HA-MRSA5 target. FIG. 20Ishows full LATE-PCR multiplex endpoint ratio fluorescence showingHA-MRSA7 target. FIG. 20J shows full LATE-PCR multiplex endpoint ratiofluorescence showing CA-MRSA2 target. FIG. 20K shows full LATE-PCRmultiplex endpoint ratio fluorescence showing CA-MRSA2 target.

FIG. 21 shows results from an exemplary MRSA type 3 assay fromExample 1. FIG. 21A shows full LATE-PCR multiplex at 3 endpoint ratiosfluorescence showing SCCmec cassette targets. FIG. 21B shows fullLATE-PCR multiplex at 3 endpoint ratios fluorescence showing SCCmeccassette targets. FIG. 21C shows full LATE-PCR multiplex endpoint ratiosfluorescence showing vanA and femA-SA targets. FIG. 21D shows fullLATE-PCR multiplex endpoint ratios fluorescence showing lukF-PVL andfemA-SE targets. FIG. 21E shows full LATE-PCR multiplex endpoint ratiosfluorescence showing internal control and mecA targets.

FIG. 22 shows an exemplary “MRSA 4” type assay configuration.

DEFINITIONS

As used herein, the phrase “probe hybridization sequence” is used isreference to a particular target sequence and a particular probe, and itis the sequence in the target sequence that hybridizes to the particularprobe. The probe may be fully or partially complementary to the targetsequence over the length of the probe hybridization sequence. However,the 5′ and 3′ terminal bases in the probe hybridization sequence areexactly complementary the probe (i.e., there are not mis-matches at the5′ and 3′ terminal ends of the probe hybridization sequence).

A “molecular beacon probe” is a single-stranded oligonucleotide,typically 25 to 35 bases-long, in which the bases on the 3′ and 5′ endsare complementary forming a “stem,” typically for 5 to 8 base pairs. Incertain embodiments, the molecular beacons employed have stems that areexactly 2 or 3 base pairs in length. A molecular beacon probe forms ahairpin structure at temperatures at and below those used to anneal theprimers to the template (typically below about 60° C.). Thedouble-helical stem of the hairpin brings a fluorophore (or other label)attached to the 5′ end of the probe very close to a quencher attached tothe 3′ end of the probe. The probe does not fluoresce (or otherwiseprovide a signal) in this conformation. If a probe is heated above thetemperature needed to melt the double stranded stem apart, or the probeis allowed to hybridize to a target oligonucleotide that iscomplementary to the sequence within the single-strand loop of theprobe, the fluorophore and the quencher are separated, and thefluorophore fluoresces in the resulting conformation. Therefore, in aseries of PCR cycles the strength of the fluorescent signal increases inproportion to the amount of the beacon hybridized to the amplicon, whenthe signal is read at the annealing temperature. Molecular beacons withdifferent loop sequences can be conjugated to different fluorophores inorder to monitor increases in amplicons that differ by as little as onebase (Tyagi, S. and Kramer, F. R. (1996), Nat. Biotech. 14:303 308;Tyagi, S. et al., (1998), Nat. Biotech. 16: 49 53; Kostrikis, L. G. etal., (1998), Science 279: 1228 1229; all of which are hereinincorporated by reference).

As used herein, the term “amplicon” refers to a nucleic acid generatedusing primer pairs, such as those described herein. The amplicon istypically single-stranded DNA (e.g., the result of asymmetricamplification), however, it may be RNA.

The term “amplifying” or “amplification” in the context of nucleic acidsrefers to the production of multiple copies of a polynucleotide, or aportion of the polynucleotide, typically starting from a small amount ofthe polynucleotide (e.g., a single polynucleotide molecule), where theamplification products or amplicons are generally detectable.Amplification of polynucleotides encompasses a variety of chemical andenzymatic processes. The generation of multiple DNA copies from one or afew copies of a target or template DNA molecule during a polymerasechain reaction (PCR) or a ligase chain reaction (LCR) are forms ofamplification. In certain embodiments, the type of amplification isasymmetric PCR (e.g., LATE-PCR) which is described in, for example, U.S.Pat. No. 7,198,897 and Pierce et al., PNAS, 2005, 102(24):8609-8614,both of which are herein incorporated by reference in their entireties.In particular embodiments, LATE-PCR is employed using multiple end-pointtemperature detection (see, e.g., U.S. Pat. Pub. 2006/0177841 andSanchez et al., BMC Biotechnology, 2006, 6:44, pages 1-14, both of whichare herein incorporated by reference in their entireties).

As used herein, the terms “complementary” or “complementarity” are usedin reference to polynucleotides (i.e., a sequence of nucleotides)related by the base-pairing rules. For example, the sequence“5′-A-G-T-3′,” is complementary to the sequence “3′-T-C-A-5′.”Complementarity may be “partial,” in which only some of the nucleicacids' bases are matched according to the base pairing rules. Or, theremay be “complete” or “total” complementarity between the nucleic acids.The degree of complementarity between nucleic acid strands hassignificant effects on the efficiency and strength of hybridizationbetween nucleic acid strands. This may be importance in amplificationreactions, as well as detection methods that depend upon binding betweennucleic acids.

The terms “homology,” “homologous” and “sequence identity” refer to adegree of identity. There may be partial homology or complete homology.A partially homologous sequence is one that is less than 100% identicalto another sequence. Determination of sequence identity is described inthe following example: a primer 20 nucleobases in length which isotherwise identical to another 20 nucleobase primer but having twonon-identical residues has 18 of 20 identical residues (18/20=0.9 or 90%sequence identity). In another example, a primer 15 nucleobases inlength having all residues identical to a 15 nucleobase segment of aprimer 20 nucleobases in length would have 15/20=0.75 or 75% sequenceidentity with the 20 nucleobase primer. Sequence identity may alsoencompass alternate or “modified” nucleobases that perform in afunctionally similar manner to the regular nucleobases adenine, thymine,guanine and cytosine with respect to hybridization and primer extensionin amplification reactions. In a non-limiting example, if the 5-propynylpyrimidines propyne C and/or propyne T replace one or more C or Tresidues in one primer which is otherwise identical to another primer insequence and length, the two primers will have 100% sequence identitywith each other. In another non-limiting example, Inosine (I) may beused as a replacement for G or T and effectively hybridize to C, A or U(uracil). Thus, if inosine replaces one or more C, A or U residues inone primer which is otherwise identical to another primer in sequenceand length, the two primers will have 100% sequence identity with eachother. Other such modified or universal bases may exist which wouldperform in a functionally similar manner for hybridization andamplification reactions and will be understood to fall within thisdefinition of sequence identity.

As used herein, the term “hybridization” or “hybridize” is used inreference to the pairing of complementary nucleic acids. Hybridizationand the strength of hybridization (i.e., the strength of the associationbetween the nucleic acids) is influenced by such factors as the degreeof complementary between the nucleic acids, stringency of the conditionsinvolved, the melting temperature (T_(M)) of the formed hybrid, and theG:C ratio within the nucleic acids. A single molecule that containspairing of complementary nucleic acids within its structure is said tobe “self-hybridized.” An extensive guide to nucleic hybridization may befound in Tijssen, Laboratory Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Acid Probes, part I, chapter 2,“Overview of principles of hybridization and the strategy of nucleicacid probe assays,” Elsevier (1993), which is incorporated by referencein its entirety.

As used herein, the term “primer” refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, that is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product that is complementary to a nucleic acid strand isinduced (e.g., in the presence of nucleotides and an inducing agent suchas a biocatalyst (e.g., a DNA polymerase or the like) and at a suitabletemperature and pH). The primer is typically single stranded for maximumefficiency in amplification, but may alternatively be double stranded.If double stranded, the primer is generally first treated to separateits strands before being used to prepare extension products. In someembodiments, the primer is an oligodeoxyribonucleotide. The primer issufficiently long to prime the synthesis of extension products in thepresence of the inducing agent. The exact lengths of the primers willdepend on many factors, including temperature, source of primer and theuse of the method. In certain embodiments, the primer is a captureprimer.

In some embodiments, the oligonucleotide primer pairs described hereincan be purified. As used herein, “purified oligonucleotide primer pair,”“purified primer pair,” or “purified” means an oligonucleotide primerpair that is chemically-synthesized to have a specific sequence and aspecific number of linked nucleosides. This term is meant to explicitlyexclude nucleotides that are generated at random to yield a mixture ofseveral compounds of the same length each with randomly generatedsequence. As used herein, the term “purified” or “to purify” refers tothe removal of one or more components (e.g., contaminants) from asample.

As used herein, the term “nucleic acid molecule” refers to any nucleicacid containing molecule, including but not limited to, DNA or RNA. Theterm encompasses sequences that include any of the known base analogs ofDNA and RNA including, but not limited to, 4 acetylcytosine,8-hydroxy-N6-methyladenosine, aziridinylcytosine, pseudoisocytosine,5-(carboxyhydroxyl-methyl) uracil, 5-fluorouracil, 5-bromouracil,5-carboxymethylaminomethyl-2-thiouracil,5-carboxymethyl-aminomethyluracil, dihydrouracil, inosine,N6-isopentenyladenine, 1-methyladenine, 1-methylpseudo-uracil,1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2-methyladenine,2-methylguanine, 3-methyl-cytosine, 5-methylcytosine, N6-methyladenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxy-amino-methyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarbonylmethyluracil, 5-methoxyuracil,2-methylthio-N-isopentenyladenine, uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, oxybutoxosine, pseudouracil, queosine,2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,5-methyluracil, N-uracil-5-oxyacetic acid methylester,uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and2,6-diaminopurine.

As used herein, the term “nucleobase” is synonymous with other terms inuse in the art including “nucleotide,” “deoxynucleotide,” “nucleotideresidue,” “deoxynucleotide residue,” “nucleotide triphosphate (NTP),” ordeoxynucleotide triphosphate (dNTP). As is used herein, a nucleobaseincludes natural and modified residues, as described herein.

An “oligonucleotide” refers to a nucleic acid that includes at least twonucleic acid monomer units (e.g., nucleotides), typically more thanthree monomer units, and more typically greater than ten monomer units.The exact size of an oligonucleotide generally depends on variousfactors, including the ultimate function or use of the oligonucleotide.To further illustrate, oligonucleotides are typically less than 200residues long (e.g., between 15 and 100), however, as used herein, theterm is also intended to encompass longer polynucleotide chains.Oligonucleotides are often referred to by their length. For example a 24residue oligonucleotide is referred to as a “24-mer”. Typically, thenucleoside monomers are linked by phosphodiester bonds or analogsthereof, including phosphorothioate, phosphorodithioate,phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate,phosphoranilidate, phosphoramidate, and the like, including associatedcounterions, e.g., H⁺, NH₄ ⁺, Na⁺, and the like, if such counterions arepresent. Further, oligonucleotides are typically single-stranded.Oligonucleotides are optionally prepared by any suitable method,including, but not limited to, isolation of an existing or naturalsequence, DNA replication or amplification, reverse transcription,cloning and restriction digestion of appropriate sequences, or directchemical synthesis by a method such as the phosphotriester method ofNarang et al. (1979) Meth Enzymol. 68: 90-99; the phosphodiester methodof Brown et al. (1979) Meth Enzymol. 68: 109-151; thediethylphosphoramidite method of Beaucage et al. (1981) TetrahedronLett. 22: 1859-1862; the triester method of Matteucci et al. (1981) J AmChem Soc. 103:3185-3191; automated synthesis methods; or the solidsupport method of U.S. Pat. No. 4,458,066, entitled “PROCESS FORPREPARING POLYNUCLEOTIDES,” issued Jul. 3, 1984 to Caruthers et al., orother methods known to those skilled in the art. All of these referencesare incorporated by reference in their entireties.

As used herein a “sample” refers to anything capable of being analyzedby the methods provided herein. In some embodiments, the samplecomprises or is suspected to comprise one or more nucleic acids capableof analysis by the methods. Preferably, the samples comprise nucleicacids (e.g., DNA, RNA, cDNAs, etc.) from one or more bioagents, such asMRSA. Samples can include, for example, blood, saliva, urine, feces,anorectal swabs, vaginal swabs, cervical swabs, and the like. In someembodiments, the samples are “mixture” samples, which comprise nucleicacids from more than one subject or individual. In some embodiments, themethods provided herein comprise purifying the sample or purifying thenucleic acid(s) from the sample. In some embodiments, the sample ispurified nucleic acid.

A “sequence” of a biopolymer refers to the order and identity of monomerunits (e.g., nucleotides, etc.) in the biopolymer. The sequence (e.g.,base sequence) of a nucleic acid is typically read in the 5′ to 3′direction.

The term “label” as used herein refers to any atom or molecule that canbe used to provide a detectable (preferably quantifiable) effect, andthat can be attached to a nucleic acid or protein. Labels include butare not limited to dyes; radiolabels such as ³²P; binding moieties suchas biotin; haptens such as digoxygenin; luminogenic, phosphorescent orfluorogenic moieties; and fluorescent dyes alone or in combination withmoieties that can suppress (“quench”) or shift emission spectra byfluorescence resonance energy transfer (FRET). FRET is adistance-dependent interaction between the electronic excited states oftwo molecules (e.g., two dye molecules, or a dye molecule and anon-fluorescing quencher molecule) in which excitation is transferredfrom a donor molecule to an acceptor molecule without emission of aphoton. (Stryer et al., 1978, Ann. Rev. Biochem., 47:819; Selvin, 1995,Methods Enzymol., 246:300, each incorporated herein by reference in itsentirety). As used herein, the term “donor” refers to a fluorophore thatabsorbs at a first wavelength and emits at a second, longer wavelength.The term “acceptor” refers to a moiety such as a fluorophore,chromophore, or quencher that has an absorption spectrum that overlapsthe donor's emission spectrum, and that is able to absorb some or mostof the emitted energy from the donor when it is near the donor group(typically between 1-100 nm). If the acceptor is a fluorophore, itgenerally then re-emits at a third, still longer wavelength; if it is achromophore or quencher, it then releases the energy absorbed from thedonor without emitting a photon. In some embodiments, changes indetectable emission from a donor dye (e.g. when an acceptor moiety isnear or distant) are detected. In some embodiments, changes indetectable emission from an acceptor dye are detected. In someembodiments, the emission spectrum of the acceptor dye is distinct fromthe emission spectrum of the donor dye such that emissions from the dyescan be differentiated (e.g., spectrally resolved) from each other.

Labels may provide signals detectable by fluorescence (e.g., simplefluorescence, FRET, time-resolved fluorescence, fluorescencepolarization, etc.), radioactivity, colorimetry, gravimetry, X-raydiffraction or absorption, magnetism, enzymatic activity,characteristics of mass or behavior affected by mass (e.g., MALDItime-of-flight mass spectrometry), and the like. A label may be acharged moiety (positive or negative charge) or alternatively, may becharge neutral.

“T_(M),” or “melting temperature,” of an oligonucleotide describes thetemperature (in degrees Celsius) at which 50% of the molecules in apopulation of a single-stranded oligonucleotide are hybridized to theircomplementary sequence and 50% of the molecules in the population arenot-hybridized to said complementary sequence. The T_(M) of a primer orprobe can be determined empirically by means of a melting curve. In somecases it can also be calculated. For the design of symmetric andasymmetric PCR primer pairs, balanced T_(M)'s are generally calculatedby one of the three methods discussed earlier, that is, the “% GC”, orthe “2(A+T) plus 4 (G+C)”, or “Nearest Neighbor” formula at some chosenset of conditions of monovalent salt concentration and primerconcentration. In the case of Nearest Neighbor calculations the T_(M)'sof both primers will depend on the concentrations chosen for use incalculation or measurement, the difference between the T_(M)'s of thetwo primers will not change substantially as long as the primerconcentrations are equimolar, as they normally are with respect to PCRprimer measurements and calculations. T_(M)[1] describes the calculatedT_(M) of a PCR primer at particular standard conditions of 1 micromolar(1 uM=10⁻⁶M) primer concentration, and 0.07 molar monovalent cations. Inthis application, unless otherwise stated, T_(M)[1] is calculated usingNearest Neighbor formula, T_(M)=ΔH/(ΔS+R ln(C/2))−273.15+12 log [M].This formula is based on the published formula (Le Novere, N. (2001),“MELTING, Computing the Melting Temperature of Nucleic Acid Duplex,”Bioinformatics 17: 1226 7). ΔH is the enthalpy and ΔS is the entropy(both ΔH and ΔS calculations are based on Allawi and SantaLucia, 1997),C is the concentration of the oligonucleotide (10⁻⁶M), R is theuniversal gas constant, and [M] is the molar concentration of monovalentcations (0.07). According to this formula the nucleotide basecomposition of the oligonucleotide (contained in the terms ΔH and ΔS),the salt concentration, and the concentration of the oligonucleotide(contained in the term C) influence the T_(M). In general foroligonucleotides of the same length, the T_(M) increases as thepercentage of guanine and cytosine bases of the oligonucleotideincreases, but the T_(M) decreases as the concentration of theoligonucleotide decreases. In the case of a primer with nucleotidesother than A, T, C and G or with covalent modification, T_(M)[1] ismeasured empirically by hybridization melting analysis as known in theart.

“T_(M)[0]” means the T_(M) of a PCR primer or probe at the start of aPCR amplification taking into account its starting concentration,length, and composition. Unless otherwise stated, T_(M)[0] is thecalculated T_(M) of a PCR primer at the actual starting concentration ofthat primer in the reaction mixture, under assumed standard conditionsof 0.07 M monovalent cations and the presence of a vast excessconcentration of a target oligonucleotide having a sequencecomplementary to that of the primer. In instances where a targetsequence is not fully complementary to a primer it is important toconsider not only the T_(M)[0] of the primer against its complements butalso the concentration-adjusted melting point of the imperfect hybridformed between the primer and the target. In this application, T_(M)[0]for a primer is calculated using the Nearest Neighbor formula andconditions stated in the previous paragraph, but using the actualstarting micromolar concentration of the primer. In the case of a primerwith nucleotides other than A, T, C and G or with covalent modification,T_(M)[0] is measured empirically by hybridization melting analysis.

As used herein superscript X refers to the Excess Primer, superscript Lrefers to the Limiting Primer, superscript A refers to the amplicon, andsuperscript P refers to the probe.

T_(M) ^(A) means the melting temperature of an amplicon, either adouble-stranded amplicon or a single-stranded amplicon hybridized to itscomplement. In this application, unless otherwise stated, the meltingpoint of an amplicon, or T_(M) ^(A), refers to the T_(M) calculated bythe following % GC formula: T_(M) ^(A)=81.5+0.41(% G+% C)−500/L+16.6 log[M]/(1+0.7 [M]), where L is the length in nucleotides and [M] is themolar concentration of monovalent cations.

T_(M)[0]^(P) refers to the concentration-adjusted melting temperature ofthe probe to its target, or the portion of probe that actually iscomplementary to the target sequence (e.g., the loop sequence of amolecular beacon probe). In the case of most linear probes, T_(M)[0]^(P)is calculated using the Nearest Neighbor formula given above, as forT_(M)[0], or preferably is measured empirically. In the case ofmolecular beacons, a rough estimate of T_(M)[0]^(P) can be calculatedusing commercially available computer programs that utilize the % GCmethod, see Marras, S. A. et al. (1999) “Multiplex Detection ofSingle-Nucleotide Variations Using Molecular Beacons,” Genet. Anal.14:151 156, or using the Nearest Neighbor formula, or preferably ismeasured empirically. In the case of probes having non-conventionalbases and for double-stranded probes, T_(M)[0]^(P) is determinedempirically.

C_(T) means threshold cycle and signifies the cycle of a real-time PCRamplification assay in which signal from a reporter indicative ofamplicons generation first becomes detectable above background. Becauseempirically measured background levels can be slightly variable, it isstandard practice to measure the C_(T) at the point in the reaction whenthe signal reaches 10 standard deviations above the background levelaveraged over the 5-10 preceding thermal cycles.

DETAILED DESCRIPTION

Provided herein are methods, kits, and compositions related to nucleicacid detection assays that allow discrimination of multiple targetsequences with a single probe. In particular, provided herein aremethods kits, and compositions that include single-probe target sequencediscrimination where different target amplicons may have identical probehybridization sequences by employing multiple temperature end-pointsignal probe detection. Also provided herein are methods, kits, andcompositions for distinguishing between two or more target ampliconsusing multiple-temperature end-point probe detection. In certainembodiments, asymmetric PCR amplification methods are employed (e.g.,LATE-PCR amplification).

Work conducted during the development of embodiments described hereindemonstrated that polymorphic nucleic acid targets could be detectedwith a single probe, even where the probe hybridization site on two ormore polymorphic targets was identical. This finding is surprising asthe art has relied on the use of different probes for different targetsequences, or the use of single probe where the probe hybridization siteis different (see, e.g., Sanchez et al., BMC Biotechnology, 2006, 6:44,pages 1-14, and U.S. Pat. Pub. 2006/0177841; both of which are hereinincorporated by reference). While not limited to any particularmechanism, and an understanding of the mechanism is not necessary topractice the methods, compositions, and kits provided herein, it isbelieved that targets with identical probe hybridization regions can bedistinguished based on the secondary structure of the target sequences.

While the polymorphic target sequences in embodiments of the assays haveidentical probe hybridization sites, such sequences do not have completesequence identity (e.g., amplicons from two or more target sequences donot have complete sequence identity). Base differences between differenttarget sequence amplicons may be located adjacent to the probehybridization site, e.g., immediately adjacent, or may be further awaynear the 5′ and 3′ ends. Such base differences, it has been discovered,cause a single probe to have a different melting temperature for theprobe hybridization site present on two or more target sequences. Themelting temperature differences can be employed to distinguish differenttarget sequences with a single probe, even though they have identicalprobe hybridization sites. Preferably, the melting or annealing of theprobe to different target amplicons is measured at least two differenttemperatures along a melting or binding curve such that atemperature/temperature signal ratio is generated. Different targetsequences, while potentially very similar in sequence, and identical atthe probe hybridization site for a given probe, will have different,characteristic, temperature/temperature signal ratios. Such differencesin temperature/temperature signal ratios allows such polymorphic targetsequences to be distinguished with a single labeled probe.

In certain embodiments, the assays described herein employ primer pairsto amplify target nucleic acid sequences. The methods described hereinare not limited by the type of amplification that is employed. Incertain embodiments, asymmetric PCR is employed, such as LATE-PCR.

PCR is a repeated series of steps of denaturation, or strand melting, tocreate single-stranded templates; primer annealing; and primer extensionby a thermally stable DNA polymerase such as Thermus aquaticus (Taq) DNApolymerase. A typical three-step PCR protocol (see Innis et al.,Chapter 1) may include denaturation, or strand melting, at 93-95 degreesC. for more than 5 sec, primer annealing at 55-65 degrees C. for 10-60sec, and primer extension for 15-120 sec at a temperature at which thepolymerase is highly active, for example, 72 degrees C. for Taq DNApolymerase. A typical two-step PCR protocol may differ by having thesame temperature for primer annealing as for primer extension, forexample, 60 degrees C. or 72 degrees C. For either three-step PCR ortwo-step PCR, an amplification involves cycling the reaction mixturethrough the foregoing series of steps numerous times, typically 25-40times. During the course of the reaction the times and temperatures ofindividual steps in the reaction may remain unchanged from cycle tocycle, or they may be changed at one or more points in the course of thereaction to promote efficiency or enhance selectivity. In addition tothe pair of primers and target nucleic acid a PCR reaction mixturetypically contains each of the four deoxyribonucleotide 5′ triphosphates(dNTPs) at equimolar concentrations, a thermostable polymerase, adivalent cation, and a buffering agent. A reverse transcriptase isincluded for RNA targets, unless the polymerase possesses that activity.The volume of such reactions is typically 25-100 ul. Multiple targetsequences can be amplified in the same reaction. In the case of cDNAamplification, PCR is preceded by a separate reaction for reversetranscription of RNA into cDNA, unless the polymerase used in the PCRpossesses reverse transcriptase activity. The number of cycles for aparticular PCR amplification depends on several factors including: a)the amount of the starting material, b) the efficiency of the reaction,and c) the method and sensitivity of detection or subsequent analysis ofthe product. Cycling conditions, reagent concentrations, primer design,and appropriate apparatuses for typical cyclic amplification reactionsare known (see, for example, Ausubel, F. Current Protocols in MolecularBiology (1988) Chapter 15: “The Polymerase Chain Reaction,” J. Wiley(New York, N.Y. (USA)).

In an idealized example, each strand of each amplicon molecule binds aprimer at one end and serves as a template for a subsequent round ofsynthesis. The rate of generation of primer extension products, oramplicons, is thus generally exponential, theoretically doubling duringeach cycle. The amplicons include both plus (+) and minus (−) strands,which hybridize to one another to form double strands. To differentiatetypical PCR from special variations described herein, typical PCR isreferred to as “symmetric” PCR. Symmetric PCR thus results in anexponential increase of one or more double-stranded amplicon molecules,and both strands of each amplicon accumulate in equal amounts duringeach round of replication. The efficiency of exponential amplificationvia symmetric PCR eventually declines, and the rate of ampliconaccumulation slows down and stops. Kinetic analysis of symmetric PCRreveals that reactions are composed of: a) an undetected amplificationphase (initial cycles) during which both strands of the target sequenceincrease exponentially, but the amount of the product thus faraccumulated is below the detectable level for the particular method ofdetection in use; b) a detected amplification phase (additional cycles)during which both strands of the target sequence continue to increase inparallel and the amount of the product is detectable; c) a plateau phase(terminal cycles) during which synthesis of both strands of the amplicongradually stops and the amount of product no longer increases. Symmetricreactions slow down and stop because the increasing concentrations ofcomplementary amplicon strands hybridize to each other (reanneal), andthis out-competes the ability of the separate primers to hybridize totheir respective target strands. Typically reactions are run long enoughto guarantee accumulation of a detectable amount of product, withoutregard to the exact number of cycles needed to accomplish that purpose.

A technique that has found limited use for making single-stranded DNAdirectly in a PCR reaction is “asymmetric PCR.” Gyllensten and Erlich,“Generation of Single-Stranded DNA by the polymerase chain reaction andits application to direct sequencing of the HLA-DQA Locus,” Proc. Natl.Acad. Sci. (USA) 85: 7652 7656 (1988); Gyllensten, U. B. and Erlich, H.A. (1991) “Methods for generating single stranded DNA by the polymerasechain reaction” U.S. Pat. No. 5,066,584, Nov. 19, 1991; all of which areherein incorporated by reference in their entireties. Asymmetric PCRdiffers from symmetric PCR in that one of the primers is added inlimiting amount, typically 1/100th to ⅕th of the concentration of theother primer. Double-stranded amplicon accumulates during the earlytemperature cycles, as in symmetric PCR, but one primer is depleted,typically after 15-25 PCR cycles, depending on the number of startingtemplates. Linear amplification of one strand takes place duringsubsequent cycles utilizing the undepleted primer. Primers used inasymmetric PCR reactions reported in the literature, including theGyllensten patent, are often the same primers for use in symmetric PCR.Poddar (Poddar, S. (2000) “Symmetric vs. Asymmetric PCR and MolecularBeacon Probe in the Detection of a Target Gene of Adenovirus,” Mol. CellProbes 14: 25 32 compared symmetric and asymmetric PCR for amplifying anadenovirus substrate by an end-point assay that included 40 thermalcycles. He reported that a primers ratio of 50:1 was optimal and thatasymmetric PCR assays had better sensitivity that, however, droppedsignificantly for dilute substrate solutions that presumably containedlower numbers of target molecules. In some embodiments, asymmetric PCRis used with embodiments of the assays described herein.

In certain embodiments, an amplification method is used that is known as“Linear-After-The Exponential PCR” or, for short, “LATE-PCR.” LATE-PCRis a non-symmetric PCR method; that is, it utilizes unequalconcentrations of primers and yields single-stranded primer-extensionproducts, or amplicons. LATE-PCR includes innovations in primer design,in temperature cycling profiles, and in hybridization probe design.Being a type of PCR process, LATE-PCR utilizes the basic steps of strandmelting, primer annealing, and primer extension by a DNA polymerasecaused or enabled to occur repeatedly by a series of temperature cycles.In the early cycles of a LATE-PCR amplification, when both primers arepresent, LATE-PCR amplification amplifies both strands of a targetsequence exponentially, as occurs in conventional symmetric PCR.LATE-PCR then switches to synthesis of only one strand of the targetsequence for additional cycles of amplification. In certain real-timeLATE-PCR assays, the limiting primer is exhausted within a few cyclesafter the reaction reaches its C_(T) value, and in the certain assaysone cycle after the reaction reaches its C_(T) value. As defined above,the C_(T) value is the thermal cycle at which signal becomes detectableabove the empirically determined background level of the reaction.Whereas a symmetric PCR amplification typically reaches a plateau phaseand stops generating new amplicons by the 50th thermal cycle, LATE-PCRamplifications do not plateau and continue to generate single-strandedamplicons well beyond the 50th cycle, even through the 100th cycle.LATE-PCR amplifications and assays typically include at least 60 cycles,preferably at least 70 cycles when small (10,000 or less) numbers oftarget molecules are present at the start of amplification.

With certain exceptions, the ingredients of a reaction mixture forLATE-PCR amplification are generally the same as the ingredients of areaction mixture for a corresponding symmetric PCR amplification. Themixture typically includes each of the four deoxyribonucleotide 5′triphosphates (dNTPs) at equimolar concentrations, a thermostablepolymerase, a divalent cation, and a buffering agent. As with symmetricPCR amplifications, it may include additional ingredients, for examplereverse transcriptase for RNA targets. Non-natural dNTPs may beutilized. For instance, dUTP can be substituted for dTTP and used at 3times the concentration of the other dNTPs due to the less efficientincorporation by Taq DNA polymerase.

In certain embodiments, the starting molar concentration of one primer,the “Limiting Primer,” is less than the starting molar concentration ofthe other primer, the “Excess Primer.” The ratio of the startingconcentrations of the Excess Primer and the Limiting Primer is generallyat least 5:1, preferably at least 10:1, and more preferably at least20:1. The ratio of Excess Primer to Limiting Primer can be, for example,5:1 . . . 10:1, 15:1 . . . 20:1 . . . 25:1 . . . 30:1 . . . 35:1 . . .40:1 . . . 45:1 . . . 50:1 . . . 55:1 . . . 60:1 . . . 65:1 . . . 70:1 .. . 75:1 . . . 80:1 . . . 85:1 . . . 90:1 . . . 95:1 . . . or 100:1 . .. 1000:1 . . . or more. Primer length and sequence are adjusted ormodified, preferably at the 5′ end of the molecule, such that theconcentration-adjusted melting temperature of the Limiting Primer at thestart of the reaction, T_(M)[0]^(L), is greater than or equal (plus orminus 0.5 degrees C.) to the concentration-adjusted melting point of theExcess Primer at the start of the reaction, T_(M)[0]^(X). Preferably thedifference (T_(M)[0]^(L)-T_(M)[0]^(X)) is at least +3, and morepreferably the difference is at least +5 degrees C.

Amplifications and assays according to embodiments of methods describedherein can be performed with initial reaction mixtures having ranges ofconcentrations of target molecules and primers. LATE-PCR assays areparticularly suited for amplifications that utilize smallreaction-mixture volumes and relatively few molecules containing thetarget sequence, sometimes referred to as “low copy number.” WhileLATE-PCR can be used to assay samples containing large amounts oftarget, for example up to 10⁶ copies of target molecules, other rangesthat can be employed are much smaller amounts, from to 1-50,000 copies,1-10,000 copies and 1-1,000 copies. In certain embodiments, theconcentration of the Limiting Primer is from a few nanomolar (nM) up to200 nM. The Limiting Primer concentration is preferably as far towardthe low end of the range as detection sensitivity permits.

As with PCR, either symmetric or asymmetric, LATE-PCR amplificationsinclude repeated thermal cycling through the steps of strand melting,primer annealing and primer extension. Temperatures and times for thethree steps are typically, as with symmetric PCR, 93-95 degrees C. forat least 5 sec for strand melting, 55-65 degrees C. for 10-60 sec forannealing primers, and 72 degrees C. for 15-120 sec for primerextension. For 3-step PCR amplifications, primer annealing times aregenerally in the range of 10-20 sec. Variations of temperature and timefor PCR amplifications are known to persons skilled in the art and aregenerally applicable to LATE-PCR as well. For example, so-called“2-step” PCR, in which one temperature is used for both primer annealingand primer extension, can be used for LATE-PCR. In the case of “2-step”reactions the combined annealing-extension step can be longer than 30sec, but preferably as short as possible and generally not longer that120 sec.

Design of primer pairs for use in LATE-PCR can be performed directly, aswill be explained. Alternatively, it can begin with selecting ordesigning a primer pair for symmetric PCR by known methods, followed bymodifications for LATE-PCR. In general, symmetric PCR primers aredesigned to have equal melting points at some set of standard conditionsof primers concentration and salt concentration. Symmetric PCR primersare conveniently designed and analyzed utilizing an available computerprogram. For symmetric and asymmetric PCR the standard techniques forcalculating melting temperatures (T_(M)) have been the “NearestNeighbor” method and the “2(A+T)+4(G+C)” method. As discussed above,T_(M)[1] which is the T_(M) of the primer at a standard primerconcentration of 1 uM and 0.07M salt (monovalent cations). Conversionfrom the T_(M) given by a typical computer program to T_(M)[1] generallyhas minimal effect on the relationship of the T_(M)'s of a primer pair.For the concentration-adjusted melting temperatures of primer pairs inembodiments described herein, either actual measurement or anappropriate calculation is generally required.

In practice, once a particular target sequence (for instance a sequenceflanking a mutation within a gene) has been chosen for amplification,several candidate pairs of equal T_(M) primers are designed via acomputer program such as Oligo 6.0® using the program's default values.The candidate primer pairs can then be scrutinized on the basis ofadditional criteria, such as possible primer-dimer formation, that areknown in the art to cause non-desirable primer qualities. Satisfactorypairs of candidate primers are further scrutinized using software suchas “Blast” for possible non-specific matches to DNA sequences elsewherein the known genome from the species of the target sequence (Madden, T.L. et al. (1996) “Applications of Network BLAST Server,” Meth. Enzymol.266: 131 141). Primers pairs are then compared as to their T_(M)[0]values at several different possible concentrations and ratios such thatthe primer chosen to be the Limiting Primer will have an equal orgreater T_(M)[0] relative to the primer chosen to be the Excess Primer.In addition, pairs of candidate primers are examined in relation to thesequence of the amplicon they are expected to generate. For instance,certain target sequences may contain a GC-rich sequence at one end and aless GC-rich sequence at the other end. Where that occurs, choosing theLimiting Primer sequence within sequences at the GC-rich end will assistin achieving a higher melting point for the Limiting Primer relative tothe Excess Primer, which will consists of sequences in the less GC-richend. Examination of the candidate primer pairs relative to the ampliconsequence may suggest additional or different ways of modifying thesequences of one or both members of the pair, such as deliberatelyincreasing or decreasing the length of the primer, most preferably atits 5′ end, or introducing changes in base sequences within the primerwhich deliberately cause it to mismatch with its target in smallregions. All such changes will increase or decrease the T_(M)[0] ofeither the Limiting or Excess primer.

EXAMPLES

The following Examples are presented in order to provide certainexemplary embodiments of the assays described herein and are notintended to limit the scope thereof.

Example 1 Monoplex and Multiplex Detection of Staphylococcus aureus

This Example describes various monoplex and multiplex S. aureusdetection assays using a form of asymmetric PCR called LATE-PCR, as wellas multiple temperature end point reads designed to generatetemperature/temperature signal ratios. The genes or regions that can beused to distinguish different bacteria types are shown in FIG. 1A. Theassays shown in the Example below identify both Methicillin-susceptibleStaphylococcus aureus (MSSA) and Methicillin-resistant Staphylococcusaureus (MRSA), by femA-SA, mecA, and orfX-SCCmec gene detection, as wellas differentiate Staph A from other coagulase negative Staph. Forexample, Staphylococcus epidermidis, MSSE and MRSE are distinguished viafemA-SE gene detection. In order to distinguish hospital acquiredHA-MRSA from community acquired MRSA (CA-MRSA), the PVL toxin geneslukF-PVL or lukS-PVL are employed. Vancomycin resistance in MRSA isdistinguished using the vanA gene. Table 1 shows the variousamplification targets and putative result(s).

TABLE 1 Amplification target Putative result femA-SA. MSSA femA-SA,mecA, orfX-SCCmec I-VII MRSA femA-SA, orfX-SCCmec I-VII MSSA (SCCmecI-VII inserted, mecA excised) femA-SE MSSE femA-SE, mecA MRSEfemA-SA/femA-SE MSSA vs. MSSE mixture: femA-SA, lukF-PVL CA-MSSAfemA-SA, orfX-SCCmec I-VII, mecA, CA-MRSA lukF-PVL femA-SA, orfX-SCCmecI-VII, mecA, VRSA vanA femA-SE, mecA, vanA VRSE Controls only Nobacteria

The genes of interest in the assays below, as shown in FIG. 1A, arefemA, mecA, lukF-PVL, vanA, and the SCCmec-orfX boundary. With the fullmulti-plex of all of these targets, there are 42 possible outcomes, asshown in FIG. 1B. Detection of these genes allows for a wide targetdifferentiation, for example, in a single multiplex assay. In some casesthe sequence differences of a gene are used to differentiate twodifferent distinct targets. The various gene/region targets are reviewedbelow.

The femA gene is used as the main criteria for detection anddifferentiation between Staphylococcus aureus and Staphylococcusepidermidis. The mecA gene is used to distinguishMethicillin-susceptible Staphylococcus aureus (MSSA) andMethicillin-resistant Staphylococcus aureus (MRSA). The mecA gene islocated in the SCCmec cassette and can also ‘pop-out’ of the cassetteleaving the cassette behind in the organism and rendering the bacteriaonce again susceptible to Methicillin. The lukF-PVL gene is an indicatorof a deadly toxin that can be incorporated into the S. aureus bacteriaand is usually associated with the a community acquired strain of S.aureus and not the hospital acquired type. It can be incorporated intoMSSA and MRSA. The vanA gene determines resistance to vancomycin, one ofthe last line antibiotics for the treatment of S. aureus infections. ThevanA gene is also found in other bacteria specifically enterococcifaecalis and faecium. The SCCmec cassette carries the mecA gene andtherefore when it inserted in S. aureus it confers resistance tomethicillin antibiotics. The SCCmec is inserted at the 3′ end of theorfX gene. The orfX gene is highly conserved while the SCCmec cassettesare not and have been described in the literature as having I-VIIdistinct types and sub-types to each of the main varieties. The assaydescribed in the Examples below is designed to detect each of the SCCmectypes and determine the SCCmec type present in the organism. This resultis accomplished using three different limiting primers that are locatedin the SCCmec sequence and a single excess primer and probe located inthe conserved orfX sequence. The T_(M) of the limiting primers vary from66° C. to 50° C. depending on sequence variation. The probe has a singlemismatch for several of the SCCmec types.

Material and Methods

The following materials and methods are used for the assays describedbelow. Table 2 provides the forward/limiting, reverse/excess, targetamplicon, and probe sequences used to detect the above discussedtargets. In Table 2, in the various amplicons, the excess/reverse primersequence, probe hybridization sequence, and complement of limitingprimer are all underlined. The probes in Table 2 (molecular beaconprobes) are shown with the hybridization portion of the probeunderlined, and the part of the probe that forms the “stem” of themolecular beacon non-underlined.

TABLE 2 SEQ T_(M) Name Sequence ID NO: Bases ° C. mecA ampliconCTGATTAACCCAGTACAGATCCTTTCAATCTATAGC 1 116 80.0GCATTAGAAAATAATGGCAATATTAACGCACCTCACTTATTAAAAGACACTTAATTGGCAAATCCGGTACTG CAGAACTC mecA excessCTGATTAACCCAGTACAGATCCT 2 23 65.3 primer mecA limitingGAGTTCTGCAGTACCGGATTTGCCA 3 24 68.5 primer mecA probeQuasar-AAGAGGTGCGTTAATATTGCTT-BHQ2 4 22 58.7 femA-SACGTTGTCTATACCTACATATCGATCCATATTTACCA 5 116 78.6 ampliconTATCAATACTTGAATCATGATGGCGAGATTACAGGTAATTGATAAAATGAGTAACTTAGGATTTGAACATAC TGGATTCC femA-SA excessCGTTGTCTATACCTACATATCGATCC 6 26 65.5 primer femA-SAGGAATCCAGTATGTTCAAATCCTAAGTTACTCATT 7 35 67.0 Limiting primerfemA-SA probe Cal Org-AACCTGTAATCTCGCCATT-BHQ1 8 19 58.4 femA-SETAAGAGTTGACCCATACCTTCCATATCAATATTTAA 9 120 78.3 ampliconATCAGGGAGAAATAACTGGAAATGCAGGTCATGATTGGATTTTTGATGAATTAGAGAGTTTAGGATATAAAC ACGAAGGATTCC femA-SE ExcessTAAGAGTTGACCCATACCTTCC 10 22 64.9 primer femA-SEGGAATCCTTCGTGTTTATATCCTAAACTCTCTAATT 11 40 67.9 Limiting CATC primerfemA-SE probe Cal Red- ATTTTCCAGTTATTTCTCCCTAT - 12 23 56.9 BHQ2 lukF-PVGGCAGAGATAGTTATCATTCAACTTATGGTAATGAA 13 119 79.4 ampliconATGTTTTTAGGCTCAAGACAAAGCAACTTAAATGCTGGACAAAACTTCTTGGAATATCACAAAATGCCAGTG TTATCCAGAGG lukF-PV ExcessGGCAGAGATAGTTATCATTCAACTTAT 14 27 64.4 Primer lukF-PVCCTCTGGATAACACTGGCATTTTGTGATATTCC 15 33 69.0 Limiting PrimerlukF-PV probe Cal Red-TAGTTGCTTTGTCTA-BHQ2 16 15 46.5 lukS-PVGAGGTGGCCTTTCCAATACAATATTGGTCTCAAAAC 56 158 78.6 ampliconAAATGACCCCAATGTAGATTTAATAAATTATCTACCTAAAAATAAAATAGATTCAGTAAATGTTAGTCAAACATTAGGTTATAACATAGGTGGTAATTTTAATAGTGG TCCATCAACAGGAGGTAATGGTTClukS-PV Excess GAGGTGGCCTTTCCAATAC 57 19 64.4 Primer lukS-PVGAACCATTACCTCCTGTTGATGGACCAC 58 28 69.2 Limiting primer lukS-PV probeBHQ1- TGTACCACCTATGTTATAACCTAATGCA - 59 28 58.6 FAM VanA Amplicon:GAGCAGGCTGTTTCGGGCTGTGAGGTCGGTTGTGCG 17 79 87.0 7596-7675GTATTGGGAAACAGTGCCGCGTTAGTTGTTGGCGAG GTGGACC vanA ExcessGAGCAGGCTGTTTCGG 18 16 64.5 Primer vanA LimitingGGTCCACCTCGCCAACAACTAACG 19 24 69.7 Primer vanA probeCal Org- TTAATACCGCACAAAA -BHQ1 20 16 45.6 InternalGAACATTACCTGCCATCCAAGTGTATCATATCGCAA 21 63 control DNAAACCACAATGGTCTGTTGGCTCCTTGC sequence Arabidopsis thaliana chromosome 1Excess Primer GAACATTACCTGCCATCCAA 22 20 63.4 reverse complementLimiting GCAAGGAGCCAACAGACCATTGTG 23 24 68.2 Primer Internal ctrlFAM- TATTTGCGATATGATATA -BHQ1 24 18 44.4 probe ExternalGAACATTACCTGCCATCCAAGTTAGTGGGAGCAGAC 25 60 control DNACACAATGGTCTGTTGGCTCCTTGC sequence Arabidopsis thaliana chromosome 1Excess Primer GAACATTACCTGCCATCCAA 26 20 63.4 reverse complementLimiting GCAAGGAGCCAACAGACCATTGTG 27 24 68.2 Primer External CtrlQuasar670- AACTGCTCCCACTTT - BHQ2 28 15 47.5 probe AdditionalTCATTATTCCTCAAGAAGAGATACAATCGGTCACTT 29 95 75.9 control DNATTAAGAAAGGTTTACTTGCTTATAAAATGGTTGTGA sequence CTACTAAAGATAACGAAGTTCCTExcess Primer TCATTATTCCTCAAGAAGAGATACAATCG 30 29 65.8 reverse LimitingAGGAACTTCGTTATCTTTAGTAGTCACAACCA 31 32 67.1 Primer Ctrl probeFAM (or Quasar)-ATAAACCTTTCTTAAAAT - 32 18 43.9 BHQ1 (or BHQ2)

The SCCmec amplicons, primers and probes are designed to cover thepossible SCCmec cassettes that are referenced in the literature.Although the cassettes have different numbering schemes, they arederived from the following references: “Combination of Multiplex PCRsfor Staphylococcal Cassette Chromosome mec Type Assignment: RapidIdentification System for mec, ccr, and Major Differences in JunkyardRegions”, Yoko Kondo, Teruyo Ito, Xiao Xue Ma, Shinya Watanabe, Barry N.Kreiswirth, Jerome Etienne, and Keiichi Hiramatsu, ANTIMICROBIAL AGENTSAND CHEMOTHERAPY, January 2007, p. 264-274; and “PCR for theidentification of methicillin resistant Staphylococcus aureus (MRSA)strains using a single primer pair specific for SCCmec elements and theneighboring chromosome-borne orfX”, C. Cuny and W. Witte, Clin MicrobiolInfect 2005; 11: 834-837; and “New Real-Time PCR Assay for RapidDetection of Methicillin-Resistant Staphylococcus aureus Directly fromSpecimens Containing a Mixture of Staphylococci”, A. Huletsky, R.Giroux, V. Rossbach, M. Gagnon, M. Vaillancourt, M. Bernier, F. Gagnon,K. Truchon, M. Bastien, F. J. Picard, A. van Belkum, M. Ouellette, P. H.Roy, and M. G. Bergeron1, JOURNAL OF CLINICAL MICROBIOLOGY, May 2004, p.1875-1884; all of which are herein incorporated by reference in theirentireties. Table 3 provides the forward/limiting, reverse/excess,target amplicon, and probe sequences used to detect the various SCCmeccassette targets. In Table 3, in the various target sequences, thecomplement of the excess primer sequence, probe hybridization sequence,and limiting primer sequences are all underlined. The probe in Table 3(molecular beacon probes) is shown with the hybridization portion of theprobe underlined, and the part of the probe that forms the “stem” of themolecular beacon non-underlined.

TABLE 3 SEQ T_(M), Name Sequence ID NO: Bases ° C. LimPriIIITTAGTTTTATTTATGATACGCTTCTCC 33 27 60-50 H3, H7, M3, M5 LimPriIIACCGCATCATTTATGATATGCTTCTCC 34 27 66-50 H2, H4, H5, M2, M4 LimPriIACCTCATTACTTATGATAAGCTTCTCC 35 27 66 H1, M1 ExcessTGACATTCCCACATCAAATGAT 36 22 64.4 Primer BridgeFAM- TTTCTTAAATGCTCTATACACTTGAA- 37 26 56.8/50.0 Probe BHQ1 SCCmec IACCTCATTACTTATGATAAGCTTCTCCTCGCA 38 98 80.0TAATCTTAAATGCTCTGTACACTTGTTCAATT AACACAACCCGCATCATTTGATGTGGGAATGT CASCCmec II ACCGCATCATTTATGATATGCTTCTCCACGCA 39 98 80.0TAATCTTAAATGCTCTATACACTTGCTCAATT AACACAACCCGCATCATTTGATGTGGGAATGT CASCCmec III TTAGTTTTATTTATGATACGCTTCTCCACGCA 40 98 80.0TAATCTTAAATGCTCTGTACACTTGTTCAATT AACACAACCCGCATCATTTGATGTGGGAATGT CASCCmec IV ACCGCATCATTTGTGGTACGCTTCTCCACGCA 41 98 80.0TAATCTTAAATGCTCTGTACACTTGTTCAATT AACACAACCCGCATCATTTGATGTGGGAATGT CASCCmec V ACCGCATCATTTATGATATGCTTCTCCTCGCA 42 98 80.0TAATCTTAAATGCTCTGTACACTTGTTCAATT AACACAACCCGCATCATTTGATGTGGGAATGT CASCCmec VII TTAGTTTTATTTGTGGTACGCTTCTCCACGCA 43 98 80.0TAATCTTAAATGCTCTATACACTTGTTCAATT AACACAACCCGCATCATTTGATGTGGGAATGT CA

When the limiting primers get incorporated in the LATE-PCR reactionthere is a mixture of all three products for each SCCmec cassette, butbased on the primer T_(M), one will be dominant. The targets generatedfor each SCCmec are shown below in Table 4 and the dominant target isshown in bold type.

TABLE 4 New Targets Generated by Limiting Primers SCCmec I: Lim 1ACCTCATTACTTATGATAAGCTTCTCC TCGCATAA TCTTAAATGCTCTGTACACTTG TTCAATTAACACAACCCGC ATCATTTGATGTGGGAATGTCA  (SEQ ID NO: 38) SCCmec I: Lim 2ACCGCATCATTTATGATATGCTTCTCCTCGCATAATCTTAAATGCTCTGTACACTTGTTCAATTAACACAACCCGCATCATTTGATGTGGGAATGTCA (SEQ ID NO: 44) SCCmec I: Lim 3TTAGTTTTATTTATGATACGCTTCTCCTCGCATAATCTTAAATGCTCTGTACACTTGTTCAATTAACACAACCCGCATCATTTGATGTGGGAATGTCA (SEQ ID NO: 45) SCCmec II: Lim 1ACCTCATTACTTATGATAAGCTTCTCCACGCATAATCTTAAATGCTCTATACACTTGCTCAATTAACACAACCCGCATCATTTGATGTGGGAATGTCA (SEQ ID NO: 46) SCCmec II: Lim 2ACCGCATCATTTATGATATGCTTCTCC ACGCATAA TCTTAAATGCTCTATACACTTG CTCAATTAACACAACCCGC ATCATTTGATGTGGGAATGTCA  (SEQ ID NO: 39) SCCmec II: Lim 3TTAGTTTTATTTATGATACGCTTCTCCACGCATAATCTTAAATGCTCTATACACTTGCTCAATTAACACAACCCGCATCATTTGATGTGGGAATGTCA (SEQ ID NO: 47) SCCmec III: Lim 1ACCTCATTACTTATGATAAGCTTCTCCACGCATAATCTTAAATGCTCTGTACACTTGTTCAATTAACACAACCCGCATCATTTGATGTGGGAATGTCA (SEQ ID NO: 48) SCCmec III: Lim 2ACCGCATCATTTATGATATGCTTCTCCACGCATAATCTTAAATGCTCTGTACACTTGTTCAATTAACACAACCCGCATCATTTGATGTGGGAATGTCA (SEQ ID NO: 49) SCCmec III: Lim 3TTAGTTTTATTTATGATACGCTTCTCC ACGCATAA TCTTAAATGCTCTGTACACTTG TTCAATTAACACAACCCGC ATCATTTGATGTGGGAATGTCA  (SEQ ID NO: 40) SCCmec IV: Lim 1ACCTCATTACTTATGATAAGCTTCTCCACGCATAATCTTAAATGCTCTGTACACTTGTTCAATTAACACAACCCGCATCATTTGATGTGGGAATGTCA (SEQ ID NO: 50) SCCmec IV: Lim 2ACCGCATCATTTATGATATGCTTCTCC ACGCATAA TCTTAAATGCTCTGTACACTTG TTCAATTAACACAACCCGC ATCATTTGATGTGGGAATGTCA  (SEQ ID NO: 41) SCCmec IV: Lim3TTAGTTTTATTTATGATACGCTTCTCCACGCATAATCTTAAATGCTCTGTACACTTGTTCAATTAACACAACCCGCATCATTTGATGTGGGAATGTCA (SEQ ID NO: 51) SCCmec V: Lim 1ACCTCATTACTTATGATAAGCTTCTCCTCGCATAATCTTAAATGCTCTGTACACTTGTTCAATTAACACAACCCGCATCATTTGATGTGGGAATGTCA (SEQ ID NO: 52) SCCmec V: Lim 2ACCGCATCATTTATGATATGCTTCTCC TCGCATAA TCTTAAATGCTCTGTACACTTG TTCAATTAACACAACCCGC ATCATTTGATGTGGGAATGTCA  (SEQ ID NO: 42) SCCmec V: Lim 3TTAGTTTTATTTATGATACGCTTCTCCTCGCATAATCTTAAATGCTCTGTACACTTGTTCAATTAACACAACCCGCATCATTTGATGTGGGAATGTCA (SEQ ID NO: 53) SCCmec VII: Lim 1ACCTCATTACTTATGATAAGCTTCTCCACGCATAATCTTAAATGCTCTATACACTTGTTCAATTAACACAACCCGCATCATTTGATGTGGGAATGTCA (SEQ ID NO: 54) SCCmec VII: Lim 2ACCGCATCATTTATGATATGCTTCTCCACGCATAATCTTAAATGCTCTATACACTTGTTCAATTAACACAACCCGCATCATTTGATGTGGGAATGTCA (SEQ ID NO: 55) SCCmec VII: Lim 3TTAGTTTTATTTATGATACGCTTCTCC ACGCATAA TCTTAAATGCTCTATACACTTG TTCAATTAACACAACCCGC ATCATTTGATGTGGGAATGTCA  (SEQ ID NO: 43)

Other reagents used in the assays below are as shown in Table 5.

TABLE 5 Taq Polymerase 1.25 U/25 uL Tfi- Polymerase 0.4 uL/25 uLPrimesafe #1 100 nM for Tfi- Primesafe #2 300 nM for Pt Taq EasyplexLDL22 300 nM for Tfi- Easyplex 4D22 600 nM for Tfi- Magnesium   3 mMTfi- Buffer 1X Taq Buffer 1X dNTPS 250 uM RNA Free Water Limiting Primer 50 nM Excess Primer  1 uM Probe 100 nM DNA Copy Number ~3000/25 uL

Exemplary Monoplex Assay

To describe how the 1-plex assays are performed, the protocol for thefemA assay is described below, beginning with source of materials inTable 6.

TABLE 6 Materials Nuclease free water Ambion PCR buffer Invitrogen MgCl2(Tfi kit) Invitrogen Lim primer (FemA_SA) Biosearch Ex primer (FemA-SA)Biosearch Probe (FemA-SA) Biosearch dNTPs Roche Pt Tfi (—) (taq)Invitrogen FemA-SA amplicon EurogentecSamples: FemA_SA amplicon (1×10⁶) (Eurogentec)10-fold dilution series of this to give 1×10⁶ with nuclease free water.

Methods

1. Label 2 eppendorf tubes ‘NTC mix’ and ‘FemA-SA mix’2. Pipette the volumes of reagents listed in the table 7 (below) labeled‘mix’ into both tubes.3. Add the stated volume of nuclease free water to both tubes.4. Vortex and spin the tubes, and in the meantime prepare the EZplex andTFi mix.5. Add the given volume of EZplex and TFI mix to the NTC and FEMA-SAtubes.6. Vortex and spin.7. Aliquot 25 μl of the NTC mix into 3 smartcycler tubes, labelled NTCand sealed.8. Transfer the NTC smartcycler tubes and the femA-SA mix into lab3.9. Spin down the smartcycler tubes and insert into smart cycler machine.10. Add given volume of Amplicon/ to FemA-SA mix, centrifuge and aliquot25 μl into 3 smartcyler tubes labelled femA-SA.11. Spin down these tubes and insert into the SmartCyler block.

TABLE 7 Components of MRSA 1-plex assay (femA). working water stock[final] μl/25 μl 1x 5x 10x Nuclease free water n/a n/a 13 13 65 130Total 13 13 65 130 Mix PCR buffer 5X 1 5 5 25 50 MgCl₂ (Tfi kit)  50 mM 3 nM 1.5 1.5 7.5 15 Lim primer (FemA_SA) 10 μM  50 nM 0.125 0.125 0.6251.25 Ex primer (FemA-SA) 100 μM  1000 nM  0.25 0.25 1.25 2.5 Probe(FemA-SA) 10 μM 100 nM 0.25 0.25 1.25 2.5 dNTPs  10 mM 250 μM 0.6250.625 3.125 6.25 Total 7.75 7.75 38.75 77.5 mix 4d22 and tfi togetherfirst then add to mix TFi Pt Tfi (—) (taq) 5 U/μl 2 U/25 μl 0.25 0.251.25 2.5 4d22 ezplex 0 0 0 0 Total 0.25 0.25 1.25 2.5 DNA 4 4 20 40Total 4 4 20 40 subtotal 25 25 125 250 NTC MSSA water 85 65 mix 38.7538.75 tfi- 1.25 1.25 dna 0 20 125 12512. Thermocycle using the following profile:

a) 95° C., 3 min

b) 50 cycles of:

i. 95° C., 10 s

ii. 58° C., 15 s

iii. 68° C., 30 s

c) End-point detection:

i. 70° C., read for 30 s

ii. 50° C., read for 30 s

iii. 35° C., read for 30 s

d) Anneal curve detection (using instrument software, otherwise 30 sread at each degree)

i. 95° C. to 30° C.

e) Melt curve detection (using instrument software, otherwise 30 s readat each degree)

i. 30° C. to 95° C.

Analysis of Resulting Data can be Conducted as Follows for the ExemplaryFema Monoplex and Other Monoplex Targets: a) End-Point Detection.

End-point detection method/algorithm can be employed on automatedinstruments. Exemplary raw fluorescence data is shown in FIG. 2A.Normalized data to 70 C F/F70 is shown in FIG. 2B. Calculated end-pointdouble ratio [F35/F70]/[F50/F70] is shown in FIG. 2C.

b) Anneal Curve Detection.

This method is performed to help optimize the end-point detectionmethod/algorithm. The anneal curve method also allows detection ofproducts (potentially non-specific) which may be formed during PCR. Theanneal raw fluorescence data is shown in FIG. 3A, the normalized annealfluorescence data to F70° C. is shown in FIG. 3B, and the calculatedanneal ratio (double ratio): [F35/F70]/[F47/F70], is shown in FIG. 3C.

c) Melt Curve Detection

Similar to the anneal curve method, melt curve detection is performed tohelp optimize the end-point detection method/algorithm. The melt curvemethod also allows detection of products (potentially non-specific)which may be formed during PCR. The melt raw fluorescence data is shownin FIG. 4A, the normalized melt melt fluorescence data to 70° C. isshown in FIG. 4B, and the calculated melt ratio (double ratio):[F35/F70]/F47/F70], is shown in FIG. 4C.

Exemplary Multiplex Detection

To describe how to perform a MRSA multiplex assay, as an example, theprotocol to perform the femA_SA, femA-SE, and mecA is described belowbeginning with a description of the materials in Table 8.

TABLE 8 Materials Nuclease free water Ambion PCR buffer Invitrogen MgCl2(Tfi kit) Invitrogen Lim primer (FemA_SA) Biosearch Ex primer (FemA-SA)Biosearch Probe (FemA-SA) Biosearch Lim primer (FemA_SE) Biosearch Exprimer (FemA-SE) Biosearch Probe (FemA-SE) Biosearch Lim primer (mecA)Biosearch Ex primer (mecA) Biosearch Probe (mecA) Biosearch dNTPs RochePt Tfi (—) (taq Invitrogen

Samples

FemA_SA amplicon (1×10¹²) dilute to give 1×10⁶ with NFW.FemA_SE amplicon (1×10¹²) dilute to give 1×10⁶ with NFW.MecA amplicon (1×10¹²) dilute to give 1×10⁶ with NFW.

Methods

1. Label 4 eppendorf tubes ‘NTC mix’ and ‘assay mix’2. Pipette the volumes of reagents listed in Table 9 (below) labeled‘mix’ into both tubes3. Add the stated volume of nuclease free water to both tubes.4. Vortex and spin the tubes, and in the meantime prepare the EZplex andTFi mix,5. Add the given volume of EZplex and TFI mix to the NTC and assaytubes.6. Vortex and spin7. Aliquot 25 μl of the NTC mix into 3 smartcycler tubes, labeled NTCand seal.8. Transfer the NTC smartcycler tubes and the ‘assay mix’ into lab 39. Spin down the smartcycler tubes and insert into smart cycler machine,10. Add given volume of Amplicon/ to ‘assay mix’, centrifuge and aliquot25 μl into 3 smartcyler tubes labeled appropriately.11. Spin down these tubes and insert into the smartcycler block.12. Thermocycle using the following profile:

a) 95° C., 3 min

b) 50 cycles of:

i. 95° C., 10 s

ii. 58° C., 15 s

iii. 68° C., 30 s

c) End-point detection:

i. 70° C., read for 30 s

ii. 50° C., read for 30 s

iii. 35° C., read for 30 s

d) Anneal curve detection (using instrument software, otherwise 30 sread at each degree)

i. 95° C. to 30° C.

e) Melt curve detection (using instrument software, otherwise 30 s readat each degree)

i. 30° C. to 95° C.

TABLE 9 Components of MRSA multiplex assay (femA_SA, femA-SE, and mecA).working mix stock [final] μ/25 μl 1x 5x 10x Nuclease free water n/a n/a2.25 2.25 11.25 22.50 Total 2.25 2.25 11.25 22.50 PCR buffer 5X 1 5.005.00 25.00 50.00 MgCl2 (Tfi kit) 50 mM 3 mM 1.50 1.50 7.50 15.00 Limprimer FemA-SA 10 μM 50 mM 0.13 0.13 0.63 1.25 Lim primer FemA-SE 10 μM50 mM 0.13 0.13 0.63 1.25 Lim primer MecA 10 μM 50 mM 0.13 0.13 0.631.25 Ex primer FemA-SA 100 μM 1 μM 0.25 0.25 1.25 2.50 Ex primer FemA-SE100 μM 1 μM 0.25 0.25 1.25 2.50 Exc primer MecA 100 μM 1 μM 0.25 0.251.25 2.50 probe FemA-SA 10 μM 100 mM 0.25 0.25 1.25 2.50 probe FemA-SE10 μM 100 mM 0.25 0.25 1.25 2.50 probe MecA 10 μM 100 mM 0.25 0.25 1.252.50 dNTPs 10 mM 250 μM 0.63 0.63 3.13 6.25 Total 9 9 45 90 mix 4d22 andtfi together first then add to mix Pt Tfi (—) (taq) 5 U/μl 2 U/25 μl0.25 0.25 1.25 2.50 4D22 10 μM 600 nM 1.50 1.50 7.50 15.00 Total 1.751.75 8.75 17.50 DNA FemA-SA 4.00 4.00 20.00 40.00 DNA FemA-SE 4.00 4.0020.00 40.00 DNA MecA 4.00 4.00 20.00 40.00 Total 12.00 12.00 60.00120.00 subtotal 25.00 25.0 125.0 250.0 NTC (x5) Mplex (x10) water 71.2522.50 mix 45 90 tfi-ezplex 8.75 17.50 femA-SE dna 0 40 femA_SA dna 0 40DNA MecA 0 40 125 250

Analysis of Resulting Data can be Conducted as Follows for the ExemplaryMulti-Plex Assay and Other Multi-Plex Assays: a) End-Point Detection.

End-point detection method/algorithm can be employed on automatedinstruments. Exemplary raw fluorescence data is shown in FIG. 5A-C.Normalized data to 70 C F/F70 is shown in FIG. 6A-C. Calculatedend-point double ratios [F35/F70]/F50/F70] are shown in FIG. 7.

b) Anneal Curve Detection.

This method is performed to help optimize the end-point detectionmethod/algorithm. The anneal curve method also allows detection ofproducts (potentially non-specific) which may be formed during PCR. Theanneal raw fluorescence data is shown in FIG. 8A-B, the normalizedanneal fluorescence data to F70° C. is shown in FIG. 9A-C, and thecalculated anneal ratios (double ratios): [F35/F70]/F47/F70], are shownin FIG. 10A-C.

c) Melt Curve Detection

Similar to the anneal curve method, melt curve detection is performed tohelp optimize the end-point detection method/algorithm. The melt curvemethod also allows detection of products (potentially non-specific)which may be formed during PCR. The melt raw fluorescence data is shownin FIGS. 11A-C, the normalized melt melt fluorescence data to 70° C. isshown in FIG. 12A-C, and the calculated calculated melt ratios (doubleratios): [F35/F70]/F47/F70], are shown in FIG. 13A-C.

Exemplary Assays Exemplary MRSA 1 Assays:

The MRSA 1 multiplex assay employs all of the components except for theSCCmec cassettes. In this assay both PVL toxin genes are incorporated,lukS/F. The femA-SA/SE genes are also read in the same Cal Org channelat different probe T_(M)'s. Also, two controls are incorporated into theassay. The full assay is shown in FIG. 14.

One example of the MRSA 1 assay is as follows. Using Pt Taq and 300 nMof Primesafe #2, and the MRSA eight-plex, LATE-PCR at endpoint isperformed with the addition of the internal and external controls. Alltargets are detected. FIG. 15 A-D show exemplary experimental results.Each panel shows the results of the detecting specific bacteria. It isnoted that the Cal Org channel is not performing properly on the Bio-Radmachine where background at low temperature drops below zero. It isbelieved that Pt Taq may cut the Cal Org probes and cause fluorescencein background that the machine cannot adequately handle. Signals shownare those above background after normalization and dye backgroundsubtraction. The top panel of each figure reflects the predicted resultand the bottom panel shows the exemplary experimental result. FIG. 15Ashows full LATE-PCR multiplex results for MSSA and MSSE targets, showingonly correct predicted results. FIG. 15B shows full LATE-PCR multiplexresults for MRSA and MRSE targets, showing only correct predictedresults. FIG. 15C shows full LATE-PCR multiplex results for VRSA andVRSE targets, showing only correct predicted results. FIG. 15D showsfull LATE-PCR multiplex results for CA-MRSA and control targets, showingonly correct predicted results.

A second example of the MRSA assay is as follows. Using Tfi-, a full 8plex can be run challenging the system with MSSA, MSSE, MRSA, MRSE,VRSA, VRSE, CA-MRSA, internal control, external and internal control,and 1:2 MSSA/MSSE. Three thousand (3,000) copies of each target, and 2 uTfi- and 100 nm Primesafe 1, can be employed and annealed at 58° C. andextended at 68° C., with 100 nm probes. All targets are detected asshown in FIG. 16A-D. Fam channel shows some false positives from vanAwhich could be bleed through. Full LATE-PCR multiplex endpoint ratiofluorescence showing internal control and mecA targets in shown in FIG.16A. Full LATE-PCR multiplex endpoint ratio fluorescence showinglukF-PVL and vanA targets is shown in FIG. 16B. Full LATE-PCR multiplexendpoint ratio fluorescence showing femA-SA and femA-SE targets is shownin FIG. 16C. Full LATE-PCR multiplex endpoint ratio fluorescence showingexternal control and lukS-PVL targets is shown in FIG. 16D.

Exemplary MRSA 2 Assays:

The MRSA 2 LATE-PCR multiplex assay makes one change in that the femA-SAand femA-SE are now separately read in Cal Org and Cal Red. The vanA isnow read in the low temperature part of Cal Org. These changes allow theassay to be more quantitative for femA-SA and femA-SE. FIG. 17 shows thegeneral details for MRSA 2 assays.

One example of the MRSA 2 assays is as follows. Full MRSA 2 multiplex isrun using 3,000 copies of all targets and 100 nm of each probe with 600nm of LDL22 and 300 nm of 4D22. Results are shown in FIG. 18 A-D. In theFAM channel, the lukS is strong. The internal control is strong, and isstronger in signal when the external control was added. In the Cal Orgchannel the femA-SA all are strong, but still some scatter in rawintensities even though the ratios are extremely tight. New vanA 2primers and probe used. In the Cal Red channel the femA-SE is verystrong, much tighter and ratios very tight. The lukF is strong for allsamples for the first time. In the Quasar channel the mecA is strong,very tight and the ratios extremely tight. The external control is notpresent. No false positives are present. All analyses are done on meltdata only. Full LATE-PCR multiplex endpoint ratio fluorescence showingcontrols and lukS-PVL targets is shown in FIG. 18A. Full LATE-PCRmultiplex endpoint ratio fluorescence showing femA-SA and vanA targetsis shown in FIG. 18B. Full LATE-PCR multiplex endpoint ratiofluorescence showing lukF-PVL and femA-SE targets is shown in FIG. 18C.Full LATE-PCR multiplex endpoint ratio fluorescence showing internalcontrol and mecA targets is shown in FIG. 18D.

Exemplary MRSA 3 Assays:

The MRSA 3 assays relates to the detection of SCCmec cassettes at orfXboundary. These assays use three limiting primers (see Material andMethods above) that are not all perfect matches to the SCCmec cassetteand an excess primer and FAM probe in the orfX that are highly conservedto detect cassettes I-VII. The multiplex version of these assays thatinvolves detecting the other genes discussed above is shown in FIG. 19,with the SCCmec cassettes incorporated into the FAM channel. Except forthe addition of the SCCmec detection and the loss of the FAM control,the assay is generally the same as MRSA 2 assays.

One example of the MRSA 3 assays is as follows. This assay is challengedwith MSSA, MSSE, MRSA I-VII, VRSA I, CA-MRSA II, and external control,using 600 nM of 4D22. Results are shown in FIG. 30 A-K. All SCCmec aredetected, as well as femA-SA, femA-SE, luF-PVL, and mecA. The controlsignal was very weak, and the vanA signal does appear on top of the femAfor the VRSA sample (vanA2 probe). FIG. 20A shows full LATE-PCRmultiplex endpoint ratio fluorescence showing control targets. FIG. 20Bshows full LATE-PCR multiplex endpoint ratio fluorescence showing MSSAtarget. FIG. 20C shows full LATE-PCR multiplex endpoint ratiofluorescence showing MSSE target. FIG. 20D shows full LATE-PCR multiplexendpoint ratio fluorescence showing HA-MRSA1 target. FIG. 20E shows fullLATE-PCR multiplex endpoint ratio fluorescence showing HA-MRSA2 target.FIG. 20F shows full LATE-PCR multiplex endpoint ratio fluorescenceshowing HA-MRSA3 target. FIG. 20G shows full LATE-PCR multiplex endpointratio fluorescence showing HA-MRSA4 target. FIG. 20H shows full LATE-PCRmultiplex endpoint ratio fluorescence showing HA-MRSA5 target. FIG. 20Ishows full LATE-PCR multiplex endpoint ratio fluorescence showingHA-MRSA7 target. FIG. 20J shows full LATE-PCR multiplex endpoint ratiofluorescence showing CA-MRSA2 target. FIG. 20K shows full LATE-PCRmultiplex endpoint ratio fluorescence showing CA-MRSA2 target.

A second example of the MRSA 3 types assays is as follows. Full MRSAmultiplex run may be challenged with NTC, MSSAV, MSSEVII, MRSA I, II,III, IV, V, VII, VRSAIII, CA-MRSAIV and internal control. Every signalis strong as shown in FIG. 21 A-F. FAM channel shows all SCCmeccassettes with some distortion due to SCCmec probe binding to aninternal control target. SCCmec II, IV, III-VII, I-V are verydistinctive on FAM bind and can be further defined on FAM melt usingdifferent ratios. Cal Org shows all femA-SA to have tight ratios andclear strong signal for VRSAIII. CalRed shows strong signal for femA-SEand lukF-PVL. Quasar shows that the internal control probe also binds tothe femA at low temperature where strong signal observed for controlonly and weaker for all other signals as a contribution of control probesignal. All mecA signals are strong and specific. Data are alsodisplayed calculating multiple end point ratios at 30/50 C, 30/45 C, and30/40 C that will rigidly define the specific SCCmec product found inthe reaction within the FAM channel detection. FIG. 21A shows fullLATE-PCR multiplex at 3 endpoint ratios fluorescence showing SCCmeccassette targets. FIG. 21B shows full LATE-PCR multiplex at 3 endpointratios fluorescence showing SCCmec cassette targets. FIG. 21C shows FullLATE-PCR multiplex endpoint ratios fluorescence showing vanA and femA-SAtargets. FIG. 21D shows full LATE-PCR multiplex endpoint ratiosfluorescence showing lukF-PVL and femA-SE targets. FIG. 21E shows fullLATE-PCR multiplex endpoint ratios fluorescence showing internal controland mecA targets.

As shown above, the MRSA 3 LATE-PCR multiplex assays run well for eachtargeted gene against all challenges and shows high selectivity andsensitivity. The Easyplex 4D22 at 600 nM cleans up reactions and allowsdilution series from 10̂8 to 1 target. All SCCmec cassettes aredistinguishable at endpoint bind/melt ratio analysis. One exemplarymethod is to use endpoint bind data, normalized to 60 C for SCCmec and65 C for other dye channels, then subtract out background dyefluorescence. At this point data that are significantly above abackground limit can be called as a positive result and will providesensitivity of the assay. Then data are normalized at ratios of 30/50 C,30/45 C and 30/40 C for maximum discrimination and selectivity. Eachspecific target (femA-SA and vanA) will have a discreet ratio that isindependent of concentration in specific dye channel.

Exemplary MRSA 4 Assay

An additional assay configuration is the one shown in FIG. 22. Such anassay can be run as described above for the other MRSA assays andendpoint read at the temperatures indicated in FIG. 22.

All publications and patents mentioned in the present application areherein incorporated by reference in their entireties. Variousmodification and variation of the described methods and compositionswill be apparent to those skilled in the art without departing from thescope and spirit of the assays described herein. Although the methods,compositions, and kits have been described in connection with specificexemplary embodiments, it should be understood that the claims are notlimited to these specific embodiments. Indeed, various modifications ofthe described modes for carrying out the assays described herein areintended to be within the scope of the following claims.

1. A method for identifying the presence of polymorphic target sequencevariants in a sample, comprising: a) providing: i) a sample suspected ofcontaining: a first or second variant of a polymorphic target sequence,ii) a labeled probe, iii) a first variant temperature/temperature signalratio, iv) a second variant temperature/temperature signal ratio, v) aforward primer, and vi) a reverse primer; b) combining said sample, saidlabeled probe, said forward primer, and said reverse primer to generatea combined sample and treating said combined sample under amplificationconditions such that: a first single-stranded amplicon is generated ifsaid first variant is present, and a second single-stranded amplicon isgenerated if said second variant is present, wherein said first andsecond single-stranded amplicons each comprise the following identicalsequences: i) a probe hybridization sequence, ii) a 5′ end correspondingto the sequence of said reverse primer, and iii) a 3′ end complementaryto said forward primer; and wherein said first and secondsingle-stranded amplicons do not have complete sequence identity; c)exposing said combined sample to multiple temperatures that allow saidlabeled probe to hybridize to said probe hybridization sequence andproduce temperature-dependent signals; d) detecting saidtemperature-dependent signals at least two temperatures; e) generatingan experimental temperature/temperature signal ratio; and f) comparingsaid experimental temperature/temperature signal ratio with said firstand second variant temperature/temperature signal ratios, wherein amatch between said experimental temperature/temperature signal ratio andsaid first or second variant temperature/temperature signal ratioidentifies the presence of said first or second variant in said sample.2. The method of claim 1, wherein said forward primer comprises alimiting primer and said reverse primer comprises an excess primer,wherein said excess primer is added to said combined sample at aconcentration at least five-times that of said limiting primer, andwherein said amplification conditions comprise asymmetric PCRconditions.
 3. The method of claim 2, wherein said asymmetric PCRconditions are LATE-PCR conditions, and wherein the initial meltingtemperature of said limiting primer is higher than or equal to theinitial melting temperature of said excess primer.
 4. The method ofclaim 1, wherein each of said single-stranded amplicons comprise atleast one amplicon spacer region selected from: A) a 5′ spacer regionthat is adjacent to said 5′ end and said probe hybridization site, andB) a 3′ spacer region adjacent to said 3′ end and said probehybridization site; and wherein at least one of said 5′ and 3′ spacerregions differ in sequence between said first and second single-strandedamplicons.
 5. The method of claim 1, wherein the presence of said firstor second variant is identified in said sample by finding a matchbetween said experimental temperature/temperature signal ratio and saidfirst or second variant temperature/temperature signal ratio.
 6. Themethod of claim 1, further comprising: providing a combined-varianttemperature/temperature signal ratio, and comparing said experimentaltemperature/temperature signal ratio to said combined-varianttemperature/temperature signal ratio, wherein a match between saidexperimental temperature/temperature signal ratio and saidcombined-variant temperature/temperature signal ratio identifies thepresence of both said first and second variants in said sample.
 7. Themethod of claim 6, wherein the presence of said first and secondvariants is identified in said sample by finding a match between saidexperimental temperature/temperature signal ratio and saidcombined-variant temperature/temperature signal ratio.
 8. The method ofclaim 1, wherein a match is found when said experimentaltemperature/temperature signal ratio is within 0.4 of said first orsecond variant temperature/temperature signal ratios.
 9. The method ofclaim 1, wherein said exposing said combined sample to multipletemperatures comprises gradually cooling said combined sample such thatsaid temperature-dependent signals are binding signals.
 10. The methodof claim 1, wherein said exposing said combined sample to multipletemperatures comprises gradually heating said combined sample such thatsaid temperature-dependent signals are melting signals.
 11. The methodof claim 1, wherein said generating an experimentaltemperature/temperature signal ratio includes normalizing saidtemperature-dependent signals with a reference temperature, and whereinsaid first and second variant temperature/temperature signal ratios arenormalized with said reference temperature.
 12. The method of claim 1,wherein said labeled probe comprises a molecular beacon probe.
 13. Themethod of claim 12, wherein said molecular beacon probe comprises a stemthat is precisely two base-pairs in length.
 14. The method of claim 1,wherein said polymorphic target sequences comprises a SCCmec region fromMRSA spanning the mecA-orfX boundary, and wherein said first and secondsingle-stranded amplicons each comprise a portion of the mecA gene and aportion of the orfX region.
 15. The method of claim 14, wherein saidfirst or second variants are selected from: SCCmec I, SCCmec II, SCCmecIII, SCCmec IV, SCCmec V, and SCCmec VII.
 16. A composition comprising:a) a labeled probe; b) a forward primer; c) a reverse primer; d) firstand second single-stranded amplicons that each comprise the followingidentical sequences: i) a probe hybridization sequence, ii) a 5′ endcorresponding to the sequence of said reverse primer, and iii) a 3′ endcomplementary to said forward primer; and wherein said first and secondsingle-stranded amplicons do not have complete sequence identity.
 17. Asystem comprising: a) a labeled probe; b) a first varianttemperature/temperature signal ratio; c) a second varianttemperature/temperature signal ratio; d) a forward primer; e) a reverseprimer; f) first and second single-stranded amplicons that each comprisethe following identical sequences: i) a probe hybridization sequence,ii) a 5′ end corresponding to the sequence of said reverse primer, andiii) a 3′ end complementary to said forward primer; and wherein saidfirst and second single-stranded amplicons do not have complete sequenceidentity.
 18. A method for identifying the presence of polymorphictarget sequence variants in a sample comprising: a) providing: i) asample suspected of containing: a first or second variant of apolymorphic target sequence, ii) a labeled probe, iii) a first varianttemperature/temperature signal ratio, iv) a second varianttemperature/temperature signal ratio, v) first and second forwardprimers that differ in sequence, and vi) a reverse primer; b) combiningsaid sample, said labeled probe, said first and second forward primers,and said reverse primer to generate a combined sample and treating saidcombined sample under amplification conditions such that: a firstsingle-stranded amplicon is generated comprising a first 3′ endcomplementary to said first forward primer, and a second single-strandedamplicon is generated comprising a second 3′ end complementary to saidsecond forward primer, and wherein said first and second single-strandedamplicons each further comprise the following identical sequences: i) aprobe hybridization sequence, and ii) a 5′ end corresponding to thesequence of said reverse primer, and c) exposing said combined sample tomultiple temperatures that allow said labeled probe to hybridize to saidprobe hybridization sequence and produce temperature-dependent signals;d) detecting said temperature-dependent signals at least twotemperatures; e) generating an experimental temperature/temperaturesignal ratio; and f) comparing said experimental temperature/temperaturesignal ratio with said first and second variant temperature/temperaturesignal ratios, wherein a match between said experimentaltemperature/temperature signal ratio and said first or second varianttemperature/temperature signal ratios identifies the presence of saidfirst or second variant in said sample.
 19. The method of claim 18,wherein said first and second forward primers comprise correspondingfirst and second limiting primers, and said reverse primer comprises anexcess primer, wherein said excess primer is added to said combinedsample at a concentration at least five-times that of said first andsecond limiting primers, and wherein said amplification conditionscomprise asymmetric PCR conditions.
 20. The method of claim 18, whereinsaid first single-stranded amplicon is generated in an amount that is atleast 10-fold greater than said second single-stranded amplicon, andwherein there is a match between said experimentaltemperature/temperature signal ratio and said first varianttemperature/temperature signal ratio, thereby identifying the presenceof said first variant.
 21. The method of claim 18, wherein said secondsingle-stranded amplicon is generated in an amount that is at least10-fold greater than said first single-stranded amplicon, and whereinthere is a match between said experimental temperature/temperaturesignal ratio and said second variant temperature/temperature signalratio, thereby identifying the presence of said second variant. 22-25.(canceled)